Humanized anti-factor D antibodies and uses thereof

ABSTRACT

The invention relates to anti-Factor D antibodies, their nucleic acid and amino acid sequences, the cells and vectors that harbor these antibodies and their production and their use in the preparation of compositions and medicaments for treatment of diseases and disorders associated with excessive or uncontrolled complement activation. These antibodies are useful for diagnostics, prophylaxis and treatment of disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed under 37 CFR §1.53(b), claims thebenefit under 35 USC §119(e) to U.S. Provisional Application Ser. No.61/048,431, filed on Apr. 28, 2008 and U.S. Provisional Application Ser.No. 61/048,689 filed on Apr. 29, 2008, the contents of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The complement system plays a central role in the clearance of immunecomplexes and the immune response to infectious agents, foreignantigens, virus-infected cells and tumor cells. However, complement isalso involved in pathological inflammation and in autoimmune diseases.Therefore, inhibition of excessive or uncontrolled activation of thecomplement cascade could provide clinical benefit to patients with suchdiseases and conditions.

The complement system encompasses two distinct activation pathways,designated the classical and the alternative pathways (V. M. Holers, InClinical Immunology: Principles and Practice, ed. R. R. Rich, MosbyPress; 1996, 363-391). The classical pathway is acalcium/magnesium-dependent cascade which is normally activated by theformation of antigen-antibody complexes. The alternative pathway is amagnesium-dependent cascade which is activated by deposition andactivation of C3 on certain susceptible surfaces (e.g. cell wallpolysaccharides of yeast and bacteria, and certain biopolymermaterials). Activation of the complement pathway generates biologicallyactive fragments of complement proteins, e.g. C3a, C4a and C5aanaphylatoxins and C5b-9 membrane attack complexes (MAC), which mediateinflammatory activities involving leukocyte chemotaxis, activation ofmacrophages, neutrophils, platelets, mast cells and endothelial cells,vascular permeability, cytolysis, and tissue injury.

Factor D is a highly specific serine protease essential for activationof the alternative complement pathway. It cleaves factor B bound to C3b,generating the C3b/Bb enzyme which is the active component of thealternative pathway C3/C5 convertases. Factor D may be a suitable targetfor inhibition, since its plasma concentration in humans is very low(1.8 μg/ml), and it has been shown to be the limiting enzyme foractivation of the alternative complement pathway (P. H. Lesavre and H.J. Müller-Eberhard. J. Exp. Med., 1978; 148: 1498-1510; J. E. Volanakiset al., New Eng. J. Med., 1985; 312: 395-401).

The down-regulation of complement activation has been demonstrated to beeffective in treating several disease indications in animal models andin ex vivo studies, e.g. systemic lupus erythematosus andglomerulonephritis (Y. Wang et al., Proc. Natl. Acad. Sci.; 1996, 93:8563-8568), rheumatoid arthritis (Y. Wang et al., Proc. Natl. Acad.Sci., 1995; 92: 8955-8959), cardiopulmonary bypass and hemodialysis (C.S. Rinder, J. Clin. Invest., 1995; 96: 1564-1572), hyperacute rejectionin organ transplantation (T. J. Kroshus et al., Transplantation, 1995;60:1194-1202), myocardial infarction (J. W. Homeister et al., J.Immunol., 1993; 150: 1055-1064; H. F. Weisman et al., Science, 1990;249: 146-151), reperfusion injury (E. A. Amsterdam et al., Am. J.Physiol., 1995; 268: H448-H457), and adult respiratory distress syndrome(R. Rabinovici et al., J. Immunol., 1992; 149: 1744-1750). In addition,other inflammatory conditions and autoimmune/immune complex diseases arealso closely associated with complement activation (V. M. Holers, ibid.,B. P. Morgan. Eur. J. Clin. Invest., 1994: 24: 219-228), includingthermal injury, severe asthma, anaphylactic shock, bowel inflammation,urticaria, angioedema, vasculitis, multiple sclerosis, myastheniagravis, membranoproliferative glomerulonephritis, and Sjögren'ssyndrome.

There is a need for antibody therapeutics in the field ofcomplement-mediated disorders, and humanized anti-Factor D antibodies,and antibody variants thereof, and fragments thereof (e.g.antigen-binding fragments), of the present invention provide highaffinity antibodies useful to meet this need.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates generally to antibodiescapable of inhibiting biological activities associated with Factor D.

In one aspect, the present invention relates to humanized anti-Factor Dantibodies, having a variety of therapeutically desired characteristics.The invention includes the amino acid sequences of the HVRs of thesehumanized anti-Factor D antibodies, and their corresponding nucleic acidsequences. The invention includes the amino acid sequences of thevariable domains of the heavy and light chain of the humanizedanti-Factor D antibodies, and their corresponding nucleic acidsequences. The invention includes the amino acid sequences of the heavyand light chain of the humanized anti-Factor D antibodies, and theircorresponding nucleic acid sequences.

In one aspect, specific antibodies within the scope of this inventioninclude, without limitation humanized anti-Factor D antibodies,comprising HVRs of humanized anti-Factor D Fab clones #238, 238-1,238-2, 238-3, 238-4, 238-5, 238-6, 238-7, 238-8, 238-9, 238-10 or238-11. In one embodiment, the humanized anti-Factor D antibodiescomprise the variable domains of the heavy and/or light chains ofhumanized anti-Factor D Fab clones #238,238-1, 238-2, 238-3, 238-4,238-5, 238-6, 238-7, 238-8, 238-9, 238-10 or 238-11. In one embodiment,the humanized anti-Factor D antibodies comprise the heavy and/or lightchains of humanized anti-Factor D Fab clones #238, 238-1, 238-2, 238-3,238-4, 238-5, 238-6, 238-7, 238-8, 238-9, 238-10 or 238-11. In oneembodiment, the invention includes the humanized anti-factor D Fabclones #238, 238-1, 238-2, 238-3, 238-4, 238-5, 238-6, 238-7, 238-8,238-9, 238-10 or 238-11. In one embodiment, the invention includesantibody fragments (e.g. antigen-binding fragments) of full-lengthantibodies of humanized anti-Factor D Fab clones #238, 238-1, 238-2,238-3, 238-4, 238-5, 238-6, 238-7, 238-8, 238-9, 238-10 or 238-11. Inone embodiment, the invention includes full-length antibodies orantigen-binding fragments thereof, comprising the antigen-bindingsequences of the heavy chain and/or the light chain of humanizedanti-Factor D Fab clones #238, 238-1, 238-2, 238-3, 238-4, 238-5, 238-6,238-7, 238-8, 238-9, 238-10 or238-11. In one embodiment, suchantigen-binding sequences comprise at least one, two, or three of theHVRs of the heavy chain. In one embodiment, such antigen-bindingsequences comprise at least one, two, or three of the HVRs of the lightchain. In one embodiment, such antigen-binding sequences comprise atleast a portion or all of the heavy chain variable domain. In oneembodiment, such antigen-binding sequences comprise at least a portionor all of the light chain variable domain.

In one aspect, the present invention provides antibody fragments (e.g.antigen-binding fragments) or full-length antibodies of humanizedanti-Factor D Fab clone #111, comprising at least one modification ofthe sequence of humanized anti-Factor D Fab clone #111, wherein suchfull-length antibodies or such antigen-binding fragments comprise theantigen-binding sequences of the heavy chain and/or the light chain ofhumanized anti-Factor D Fab clone #111. In one embodiment, the antibodyfragments (e.g. antigen-binding fragments) or full-length antibodies ofhumanized anti-Factor D Fab clone #111, comprising at least onemodification of the sequence of humanized anti-Factor D Fab clone #111,comprising the antigen-binding sequences of the heavy chain and/or thelight chain of humanized anti-Factor D Fab clone #111, further bindessentially to the same epitope as humanized antibody Fab clone #111. Inone embodiment, such antigen-binding sequences comprise at least one,two or three of the HVRs of the heavy chain. In one embodiment, suchantigen-binding sequences comprise at least one, two or three of theHVRs of the light chain. In one embodiment, such antigen-bindingsequences comprise at least a portion or all of the heavy chain variabledomain. In one embodiment, such antigen-binding sequences comprise atleast a portion or all of the light chain variable domain. In oneembodiment, such modification in said antibody fragments or saidfull-length antibodies, is in the heavy chain. In one embodiment, suchmodification in said antibody fragments or said full-length antibodies,is in the light chain. In one embodiment, such modification in saidantibody fragments or said full-length antibodies, is in the heavy chainvariable domain. In one embodiment, such modification in said antibodyfragments or said full-length antibodies, is in the light chain variabledomain. In one embodiment, such modification in said antibody fragmentsor said full-length antibodies, is in at least one, two or three of theHVRs of the heavy chain. In one embodiment, such modification in saidantibody fragments or said full-length antibodies, is in at least one,two or three of the HVRs of the light chain.

In one aspect, the invention concerns antibodies of the presentinvention, or fragments thereof (e.g. antigen-binding fragments), whichbind to Factor D with a binding affinity of at least about 10⁻⁹ to10⁻¹²M.

In one aspect, the invention concerns antibodies of the presentinvention, or fragments thereof (e.g. antigen-binding fragments),wherein a Fab fragment of such antibodies inhibits a biological functionof Factor D in a Fab fragment to Factor D molar ratio of about 0.05:1(0.05) to about 10:1 (10), or about 0.09:1 (0.09) to about 8:1 (8), orabout 0.1:1 (0.1) to about 6:1 (6), or about 0.15:1 (0.15) to about 5:1(5), or about 0.19:1 (0.19) to about 4:1 (4), or about 0.2:1 (0.2) toabout 3:1 (3), or about 0.3:1 (0.3) to about 2:1 (2), or about 0.4:1(0.4) to about 1:1 (1), or about 0.5:1 (0.5) to about 1:2 (0.5), orabout 0.6:1 (0.6) to about 1:3 (0.33), or about 0.7:1 (0.7) to about 1:4(0.25), or about 0.8:1 (0.8) to about 1:5 (0.2) or about 0.9:1 (0.9) toabout 1:6 (0.17).

In one aspect, the antibodies of the present invention include human,humanized or chimeric antibodies.

In another aspect, the present invention includes antibody fragments(e.g. antigen-binding fragments) of humanized anti-Factor D antibodies.The antibody fragments of the present invention may, for example, beFab, Fab′, F(ab′)₂, scFv, (scFv)₂, dAb, complementarity determiningregion (CDR) fragments, linear antibodies, single-chain antibodymolecules, minibodies, diabodies, or multispecific antibodies formedfrom antibody fragments.

In other aspects of the invention, the present invention includescompositions comprising an antibody of the invention, or fragmentthereof (e.g. antigen-binding fragment). In another embodiment, theinvention provides cell lines and vectors encoding at least a portion ofan antibody of the invention, or fragment thereof (e.g. antigen-bindingfragment). In one aspect, the invention includes method of making,method of producing, and method of using antibodies, or fragmentsthereof (e.g. antigen-binding fragments) and compositions of theinvention. In one embodiment, the method of making an antibody of theinvention, or fragment thereof (e.g. antigen-binding fragment), whereinthe method comprises (a) culturing a host cell, for example a eukaryoticor CHO cell, comprising a vector, further comprising a polynucleotideencoding an antibody of the invention, or fragment thereof (e.g.antigen-binding fragment), under conditions suitable for expression ofthe polynucleotide encoding the antibody, or fragment thereof (e.g.antigen-binding fragment) and (b) isolating the antibody, or fragmentthereof (e.g. antigen-binding fragment).

In a still further aspect, the invention concerns a composition ofmatter comprising an antibody of the invention, or fragment thereof(e.g. antigen-binding fragment), as described herein, in combinationwith a carrier. Optionally, the carrier is a pharmaceutically acceptablecarrier.

Another aspect of the present invention is the use of these humanizedantibodies or antibody fragments thereof (e.g. antigen-bindingfragments), for the preparation of a medicament or composition for theprevention and/or treatment of disorders associated with excessive oruncontrolled complement activation. They include complement activationduring cardiopulmonary bypass operations; complement activation due toischemia-reperfusion following acute myocardial infarction, aneurysm,stroke, hemorrhagic shock, crush injury, multiple organ failure,hypobolemic shock, intestinal ischemia or other events causing ischemia.Complement activation has also been shown to be associated withinflammatory conditions such as severe burns, endotoxemia, septic shock,adult respiratory distress syndrome, hemodialysis; anaphylactic shock,severe asthma, angioedema, Crohn's disease, sickle cell anemia,poststreptococcal glomerulonephritis and pancreatitis. The disorder maybe the result of an adverse drug reaction, drug allergy, IL-2 inducedvascular leakage syndrome or radiographic contrast media allergy. Italso includes autoimmune disease such as systemic lupus erythematosus,myasthenia gravis, rheumatoid arthritis, Alzheimer's disease andmultiple sclerosis. In another embodiment, complement activation is alsoassociated with transplant rejection. In another embodiment, complementactivation is also associated with ocular diseases (all ocularconditions and diseases the pathology of which involve complement,including the classical and the alternative pathway of complement), suchas, for example, without limitation, macular degenerative disease, suchas all stages of age-related macular degeneration (AMD), including dryand wet (non-exudative and exudative) forms, diabetic retinopathy andother ischemia-related retinopathies, choroidal neovascularization(CNV), uveitis, diabetic macular edema, pathological myopia, vonHippel-Lindau disease, histoplasmosis of the eye, Central Retinal VeinOcclusion (CRVO), corneal neovascularization, and retinalneovascularization. In one example, complement-associated eye conditionsinclude age-related macular degeneration (AMD), including non-exudative(e.g intermediate dry AMD or geographic atrophy (GA)) and exudative(e.g. wet AMD (choroidal neovascularization (CNV)) AMD, diabeticretinopathy (DR), endophthalmitis and uveitis. In a further example,nonexudative AMD may include the presence of hard drusen, soft drusen,geographic atrophy and/or pigment clumping. In another example,complement-associated eye conditions include age-related maculardegeneration (AMD), including early AMD (e.g. includes multiple small toone or more non-extensive medium sized drusen), intermediate AMD (e.g.includes extensive medium drusen to one or more large drusen) andadvanced AMD (e.g. includes geographic atrophy or advanced wet AMD(CNV). In a further example, intermediate dry AMD may include largeconfluent drusen. In a further example, geographic atrophy may includephotoreceptor and/or Retinal Pigmented Epithelial (RPE) loss. In afurther example, the area of geographic atrophy may be small or largeand/or may be in the macula area or in the peripheral retina. In oneexample, the complement-associated eye condition is intermediate dryAMD. In one example, the complement-associated eye condition isgeographic atrophy. In one example, the complement-associated eyecondition is wet AMD (choroidal neovascularization (CNV)).

In another aspect, the invention provides a kit, comprising an antibodyof the invention, or fragment thereof (e.g. antigen-binding fragment).In one embodiment, the invention provides a kit, comprising an antibodyof the invention, or fragment thereof (e.g. antigen-binding fragment)and instructions for use. In one embodiment, the invention concerns akit comprising an antibody of the invention, or fragment thereof (e.g.antigen-binding fragment) and instructions for administering saidantibody, to treat a complement-associated disorder. In one embodiment,the invention provides a kit comprising a first container comprising acomposition comprising one or more one or more antibodies of theinvention, or antibody fragments thereof (e.g. antigen-bindingfragments); and a second container comprising a buffer. In oneembodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antibody of the invention, orfragment thereof (e.g. antigen-binding fragment) further comprises acarrier, which in some embodiments is pharmaceutically acceptable. Inone embodiment, a kit further comprises instructions for administeringthe composition (e.g the antibody, or antibody fragment thereof (e.g.antigen-binding fragment) to a subject. In one embodiment, a kit furthercomprises instructions for use of the kit.

In one aspect, the invention concerns an article of manufacturecontaining materials useful for the treatment, prevention and/ordiagnosis of complement-associated disorders. In one embodiment, theinvention concerns an article of manufacture, comprising: (a) acontainer; (b) a label on the container; and (c) a composition of mattercomprising an antibody, or variant thereof or fragment thereof (e.g.antigen-binding fragment), of the present invention, contained with thecontainer, wherein the label on said container indicates that thecomposition can be used for treatment, prevention and/or diagnosis ofcomplement-associated disorders.

In one aspect, the invention provides use of an anti-Factor D antibodyof the invention, or antibody fragment thereof (e.g. antigen-bindingfragment), nucleic acid of the invention, expression vector of theinvention or host cell of the invention, in the preparation of amedicament for the therapeutic and/or prophylactic treatment of adisease, such as a complement-associated eye condition. In oneembodiment, the complement-associated eye condition is selected fromage-related macular degeneration (AMD), including non-exudative (e.gintermediate dry AMD or geographic atrophy (GA)) and exudative (e.g. wetAMD (choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR),endophthalmitis and uveitis. In one example, the complement-associatedeye condition is intermediate dry AMD. In one example, thecomplement-associated eye condition is geographic atrophy. In oneexample, the complement-associated eye condition is wet AMD (choroidalneovascularization (CNV)).

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as acomplement-associated eye condition. In one embodiment, thecomplement-associated eye condition is selected from age-related maculardegeneration (AMD), including non-exudative (e.g intermediate dry AMD orgeographic atrophy (GA)) and exudative (e.g. wet AMD (choroidalneovascularization (CNV)) AMD, diabetic retinopathy (DR),endophthalmitis and uveitis. In one example, the complement-associatedeye condition is intermediate dry AMD. In one example, thecomplement-associated eye condition is geographic atrophy. In oneexample, the complement-associated eye condition is wet AMD (choroidalneovascularization (CNV)).

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as a complement-associated eye condition.In one embodiment, the complement-associated eye condition is selectedfrom age-related macular degeneration (AMD), including non-exudative(e.g intermediate dry AMD or geographic atrophy (GA)) and exudative(e.g. wet AMD (choroidal neovascularization (CNV)) AMD, diabeticretinopathy (DR), endophthalmitis and uveitis. In one example, thecomplement-associated eye condition is intermediate dry AMD. In oneexample, the complement-associated eye condition is geographic atrophy.In one example, the complement-associated eye condition is wet AMD(choroidal neovascularization (CNV)).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C shows the alignment of sequences of the variable light chaindomains for the following: humanized anti-Factor D Fab clone #111 (SEQID NO: 1), humanized anti-Factor D Fabs, 238, 238-1, 238-2, 238-3,238-4, 238-5, 238-6, 238-7, 238-8, 238-9, 238-10 and 238-11 (SEQ ID NOs:6-17, respectively) and VL Kappa I consensus sequence (SEQ ID NO: 65).Positions are numbered according to Kabat and hypervariable regions (inaccordance with Kabat+Chothia HVR definitions) are boxed (HVRs: (1)HVR-L1 identified as A1-A11 (ITSTDIDDDMN (SEQ ID NO: 30), ITSTDIDDDLN(SEQ ID NO: 31), ITSTDIDDDIN (SEQ ID NO: 32), ITSTDIDDDMA (SEQ ID NO:33) or ITSTDIDDDMQ (SEQ ID NO: 34)), (2) HVR-L2 identified as B1-B7(GGNTLRP (SEQ ID NO: 35), GGSTLRP (SEQ ID NO: 36) or GGATLRP (SEQ ID NO:37)), (3) HVR-L3 identified as C1-C9 (LQSDSLPYT (SEQ ID NO: 38)). Aminoacid changes in each humanized anti-Factor D Fab are bold anditalicized.

FIG. 2A-C shows the alignment of sequences of the variable heavy chaindomains for the following: humanized anti-Factor D Fab clone #111 (SEQID NO: 2) and humanized anti-Factor D Fabs, 238, 238-1, 238-2, 238-3,238-4, 238-5, 238-6, 238-7, 238-8, 238-9, 238-10 and 238-11 (SEQ ID NOs:18-29, respectively) and VH subgroup 7 consensus sequence (SEQ ID NO:66). Positions are numbered according to Kabat and hypervariable regions(in accordance with Kabat+Chothia HVR definitions) are boxed (HVRs: (1)HVR-H1 identified as D1-D10 (GYTFTNYGMN (SEQ ID NO: 39), (2) HVR-H2identified as E1-E19 (WINTYTGETTYADDFKG (SEQ ID NO: 40), (3) HVR-H3identified as F1-F6 (EGGVNN (SEQ ID NO: 41), EGGVAN (SEQ ID NO: 42),EGGVQN (SEQ ID NO: 43), EGGVNA (SEQ ID NO: 44) or EGGVNQ (SEQ ID NO:45)). Amino acid changes in each humanized anti-Factor D Fab are boldand italicized.

FIG. 3 shows the nucleotide sequence (SEQ ID NO: 46) of the light chainof humanized anti-Factor D Fab 238. The nucleotide sequence encodes forthe light chain of humanized anti-Factor D Fab 238 with the start andstop codon shown in bold and underlined. The codon corresponding to thefirst amino acid in FIG. 4 (SEQ ID NO: 47) is bold and italicized.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 47) of the light chainfor humanized anti-Factor D Fab 238. The amino acid sequence lacks theN-terminus signal sequence of the polypeptide encoded by SEQ ID NO: 46shown in FIG. 3. The HVR sequences are bold and italicized. Variableregions are regions not underlined while first constant domain CL1 isunderlined. Framework (FR) regions and HVR regions are shown:

FR1-LC, (SEQ ID NO: 48) FR2-LC, (SEQ ID NO: 49) FR3-LC, (SEQ ID NO: 50)FR4-LC, (SEQ ID NO: 51) HVR1-LC, (SEQ ID NO: 30, (ITSTDIDDDMN)),HVR2-LC, (SEQ ID NO: 35 (GGNTLRP)), HVR3-LC (SEQ ID NO: 38 (LQSDSLPYT))and CL1. (SEQ ID NO: 52)

FIG. 5 shows the nucleotide sequence (SEQ ID NO: 53) of the heavy chainof humanized anti-Factor D Fab 238. The nucleotide sequence encodes forthe heavy chain of humanized anti-Factor D Fab 238 with the start andstop codon shown in bold and underlined. The codon corresponding to thefirst amino acid in FIG. 6 (SEQ ID NO: 54) is bold and italicized.

FIG. 6 shows the amino acid sequence (SEQ ID NO: 54) of the heavy chainfor humanized anti-Factor D Fab 238. The amino acid sequence lacks theN-terminus signal sequence of the polypeptide encoded by SEQ ID NO: 53shown in FIG. 5. The HVR sequences are bold and italicized. Variableregions are regions not underlined while first constant domain CH1 isunderlined. Framework (FR) regions and HVR regions are shown:

FR1-HC, (SEQ ID NO: 55) FR2-HC, (SEQ ID NO: 56) FR3-HC, (SEQ ID NO: 57)FR4-HC, (SEQ ID NO: 58) HVR1-HC, (SEQ ID NO: 39 (GYTFTNYGMN)) HVR2-HC,(SEQ ID NO: 40 (WINTYTGETTYADDFKG)) HVR3-HC (SEQ ID NO: 41 (EGGVNN)) andCH1. (SEQ ID NO: 59)

FIG. 7 shows the nucleotide sequence (SEQ ID NO: 60) of the light chainof humanized anti-Factor D Fab 238-1. The nucleotide sequence encodesfor the light chain of humanized anti-Factor D Fab 238-1 with the startand stop codon shown in bold and underlined. The codon corresponding tothe first amino acid in FIG. 8 (SEQ ID NO: 61) is bold and italicized.

FIG. 8 shows the amino acid sequence (SEQ ID NO: 61) of the light chainfor humanized anti-Factor D Fab 238-1. The amino acid sequence lacks theN-terminus signal sequence of the polypeptide encoded by SEQ ID NO: 60shown in FIG. 7. The HVR sequences are bold and italicized. Variableregions are regions not underlined while first constant domain CL1 isunderlined. Framework (FR) regions and HVR regions are shown:

FR1-LC, (SEQ ID NO: 48) FR2-LC, (SEQ ID NO: 49) FR3-LC, (SEQ ID NO: 50)FR4-LC, (SEQ ID NO: 51) HVR1-LC, (SEQ ID NO: 30 (ITSTDIDDDMN)) HVR2-LC,(SEQ ID NO: 35 (GGNTLRP)) HVR3-LC (SEQ ID NO: 38 (LQSDSLPYT)) and CL1.(SEQ ID NO: 52)

FIG. 9 shows the nucleotide sequence (SEQ ID NO: 62) of the heavy chainof humanized anti-Factor D Fab 238-1. The nucleotide sequence encodesfor the heavy chain of humanized anti-Factor D Fab 238-1 with the startand stop codon shown in bold and underlined. The codon corresponding tothe first amino acid in FIG. 10 (SEQ ID NO: 63) is bold and italicized.

FIG. 10 shows the amino acid sequence (SEQ ID NO: 63) of the heavy chainfor humanized anti-Factor D Fab 238-1. The amino acid sequence lacks theN-terminus signal sequence of the polypeptide encoded by SEQ ID NO: 62shown in FIG. 9. The HVR sequences are bold and italicized. Variableregions are regions not underlined while first constant domain CH1 isunderlined. Framework (FR) regions and HVR regions are shown:

FR1-HC, (SEQ ID NO: 64) FR2-HC, (SEQ ID NO: 56) FR3-HC, (SEQ ID NO: 57)FR4-HC, (SEQ ID NO: 58) HVR1-HC, (SEQ ID NO: 39 (GYTFTNYGMN)) HVR2-HC,(SEQ ID NO: 40 (WINTYTGETTYADDFKG)) HVR3-HC (SEQ ID NO: 41 (EGGVNN)) andCH1. (SEQ ID NO: 59)

FIG. 11 shows the hemolytic assay results, showing inhibition of thealternative complement activity, for humanized anti-Factor D Fab clone#111 and humanized anti-Factor D Fabs 238 and 238-1. IC₅₀ values areshown.

FIG. 12 shows the hemolytic assay results, showing inhibition of thealternative pathway (AP) complement activity, for humanized anti-FactorD Fab 238, at three serum concentrations (9.7 nM, 16.2 nM and 26.5 nM)of Factor D. Table 3 shows the IC₅₀ (nM) and IC₉₀ (nM) values (valuesrepresent the average of three repeated experiments), corresponding tothe three serum concentrations of Factor D. The antibody to targetFactor D molar ratios are also shown in Table 3.

FIG. 13 shows the simulated duration of inhibition of the alternativepathway (AP) complement activation in a human eye using a singleintravitreal (IVT) injection of anti-Factor D Fab 238 at a 2.5 mg dose(assuming a half-life (t_(1/2)) of anti-Factor D Fab 238=11.5 days,based on interspecies scaling from the rabbit). A single IVT injectionof anti-Factor D Fab 238 was estimated to inhibit AP complementactivation in the retinal tissue for at least about 74 days and in thevitreous humor for at least about 97 days. In FIG. 13, the dashed lineshows the simulated anti-Factor D Fab 238 concentration in the vitreoushumor following intravitreal administration. In FIG. 13, the solid lineshows the simulated anti-Factor D Fab 238 concentration in the retinaltissue following intravitreal administration. The difference in theconcentration in the vitreous humor and retinal tissue is based upon anestimate of the retinal tissue partition coefficient of 20%; in otherwords, 20% of the total drug administered to the vitreous humor willhave access to the retinal tissue.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,Applicants desire that the following terms be given the particulardefinition as defined below.

The phrase “substantially identical” with respect to an antibody chainpolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, or 80%, or 90% or 95% sequence identity to the referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, or 90%, or 95% or 97% sequence identity to the reference nucleicacid sequence.

The term “identity” or “homology” shall be construed to mean thepercentage of amino acid residues in the candidate sequence that areidentical with the residue of a corresponding sequence to which it iscompared, after aligning the sequences and introducing gaps, ifnecessary to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.Sequence identity may be measured using sequence analysis software.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In one example, the antibody will be purified(1) to greater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or nonreducing conditions usingCoomassie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, “anti-human Factor D antibody” means an antibody whichspecifically binds to human Factor D in such a manner so as to inhibitor substantially reduce complement activation.

The term “Factor D” is used herein to refer to native sequence andvariant Factor D polypeptides.

A “native sequence” Factor D, is a polypeptide having the same aminoacid sequence as a Factor D polypeptide derived from nature, regardlessof its mode of preparation. Thus, native sequence Factor D can beisolated from nature or can be produced by recombinant and/or syntheticmeans. In addition to a mature Factor D protein, such as a mature humanFactor D protein (NM_(—)001928), the term “native sequence Factor D”,specifically encompasses naturally-occurring precursor forms of Factor D(e.g., an inactive preprotein, which is proteolytically cleaved toproduce the active form), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofFactor D, as well as structural conformational variants of Factor Dmolecules having the same amino acid sequence as a Factor D polypeptidederived from nature. Factor D polypeptides of non-human animals,including higher primates and non-human mammals, are specificallyincluded within this definition.

“Factor D variant” means an active Factor D polypeptide as defined belowhaving at least about 80% amino acid sequence identity to a nativesequence Factor D polypeptide, such as the native sequence human FactorD polypeptide (NM_(—)001928). Ordinarily, a Factor D variant will haveat least about 80% amino acid sequence identity, or at least about 85%amino acid sequence identity, or at least about 90% amino acid sequenceidentity, or at least about 95% amino acid sequence identity, or atleast about 98% amino acid sequence identity, or at least about 99%amino acid sequence identity with the mature human amino acid sequence(NM_(—)001928).

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in a reference Factor D sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

“Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in a reference Factor D-encoding sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes nucleic acid molecules contained in cells that ordinarilyexpress an encoded polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

An “isolated” Factor D polypeptide-encoding nucleic acid molecule is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the Factor D-encoding nucleic acid. An isolatedFactor D polypeptide-encoding nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated Factor Dpolypeptide-encoding nucleic acid molecules therefore are distinguishedfrom the encoding nucleic acid molecule(s) as they exists in naturalcells. However, an isolated Factor D-encoding nucleic acid moleculeincludes Factor D-encoding nucleic acid molecules contained in cellsthat ordinarily express Factor D where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “antagonist” is used in the broadest sense, and includes anymolecule that is capable of neutralizing, blocking, partially or fullyinhibiting, abrogating, reducing or interfering with a Factor Dbiological activity. Factor D antagonists include, without limitation,anti-Factor D antibodies, and antibody variants thereof, antigen-bindingfragments thereof, other binding polypeptides, peptides, and non-peptidesmall molecules, that bind to Factor D and are capable of neutralizing,blocking, partially or fully inhibiting, abrogating, reducing orinterfering with Factor D activities, such as the ability of Factor D toparticipate in the pathology of a complement-associated eye condition.

A “small molecule” is defined herein to have a molecular weight belowabout 600, preferable below about 1000 daltons.

“Active” or “activity” or “biological activity” in the context of aFactor D antagonist of the present invention is the ability toantagonize (partially or fully inhibit) a biological activity of FactorD. One example of a biological activity of a Factor D antagonist is theability to achieve a measurable improvement in the state, e.g.pathology, of a Factor D-associated disease or condition, such as, forexample, a complement-associated eye condition. The activity can bedetermined in in vitro or in vivo tests, including binding assays,alternative pathway hemolysis assays (e.g. assays measuring inhibitionof the alternative pathway complement activity or activation), using arelevant animal model, or human clinical trials.

The term “complement-associated disorder” is used in the broadest senseand includes disorders associated with excessive or uncontrolledcomplement activation. They include complement activation duringcardiopulmonary bypass operations; complement activation due toischemia-reperfusion following acute myocardial infarction, aneurysm,stroke, hemorrhagic shock, crush injury, multiple organ failure,hypobolemic shock, intestinal ischemia or other events causing ischemia.Complement activation has also been shown to be associated withinflammatory conditions such as severe burns, endotoxemia, septic shock,adult respiratory distress syndrome, hemodialysis; anaphylactic shock,severe asthma, angioedema, Crohn's disease, sickle cell anemia,poststreptococcal glomerulonephritis and pancreatitis. The disorder maybe the result of an adverse drug reaction, drug allergy, IL-2 inducedvascular leakage syndrome or radiographic contrast media allergy. Italso includes autoimmune disease such as systemic lupus erythematosus,myasthenia gravis, rheumatoid arthritis, Alzheimer's disease andmultiple sclerosis. Complement activation is also associated withtransplant rejection. Complement activation is also associated withocular diseases such as age-related macular degeneration, diabeticretinopathy and other ischemia-related retinopathies, choroidalneovascularization (CNV), uveitis, diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, and retinalneovascularization.

The term “complement-associated eye condition” is used in the broadestsense and includes all eye conditions the pathology of which involvescomplement, including the classical and the alternative pathways, and inparticular the alternative pathway of complement. Complement-associatedeye conditions include, without limitation, macular degenerativediseases, such as all stages of age-related macular degeneration (AMD),including dry and wet (non-exudative and exudative) forms, choroidalneovascularization (CNV), uveitis, diabetic and other ischemia-relatedretinopathies, and other intraocular neovascular diseases, such asdiabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO),corneal neovascularization, and retinal neovascularization. In oneexample, complement-associated eye conditions includes age-relatedmacular degeneration (AMD), including non-exudative (e.g. intermediatedry AMD or geographic atrophy (GA)) and exudative (e.g. wet AMD(choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR),endophthalmitis and uveitis. In a further example, nonexudative AMD mayinclude the presence of hard drusen, soft drusen, geographic atrophyand/or pigment clumping. In one example, complement-associated eyeconditions include age-related macular degeneration (AMD), includingearly AMD (e.g. includes multiple small to one or more non-extensivemedium sized drusen), intermediate AMD (e.g. includes extensive mediumdrusen to one or more large drusen) and advanced AMD (e.g. includesgeographic atrophy or advanced wet AMD (CNV). (Ferris et al., AREDSReport No. 18; Sallo et al., Eye Res., 34(3): 238-40 (2009); Jager etal., New Engl. J. Med., 359(1): 1735 (2008)). In a further example,intermediate dry AMD may include large confluent drusen. In a furtherexample, geographic atrophy may include photoreceptor and/or RetinalPigmented Epithelial (RPE) loss. In a further example, the area ofgeographic atrophy may be small or large and/or may be in the maculaarea or in the peripheral retina. In one example, complement-associatedeye condition is intermediate dry AMD. In one example,complement-associated eye condition is geographic atrophy. In oneexample, complement-associated eye condition is wet AMD (choroidalneovascularization (CNV)).

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In treatment of an immune related disease,a therapeutic agent may directly alter the magnitude of response of acomponent of the immune response, or render the disease more susceptibleto treatment by other therapeutic agents, e.g., antibiotics,antifungals, anti-inflammatory agents, chemotherapeutics, etc.

The “pathology” of a disease, such as a complement-associated eyecondition, includes all phenomena that compromise the well-being of thepatient. This includes, without limitation, abnormal or uncontrollablecell growth (neutrophilic, eosinophilic, monocytic, lymphocytic cells),antibody production, auto-antibody production, complement production,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels, suppressionor aggravation of any inflammatory or immunological response,infiltration of inflammatory cells (neutrophilic, eosinophilic,monocytic, lymphocytic) into cellular spaces, etc.

The term “mammal” as used herein refers to any animal classified as amammal, including, without limitation, humans, higher primates, domesticand farm animals, and zoo, sports or pet animals such horses, pigs,cattle, dogs, cats and ferrets, etc. In one embodiment of the invention,the mammal is a human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Therapeutically effective amount” is the amount of a “Factor Dantagonist” which is required to achieve a measurable improvement in thestate, e.g. pathology, of the target disease or condition, such as, forexample, a complement-associated eye condition.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42 C; or (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular target.However, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) also known as hypervariableregions (HVRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of variable domains arecalled the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely a adopting α-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the α-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of the targetbinding site of antibodies (see Kabat et al.). As used herein, numberingof immunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al.,(Sequences of Proteins of Immunological Interest, National Institute ofHealth, Bethesda, Md. 1987), unless otherwise indicated.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop whennumbered using the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34). The AbMhypervariable regions represent a compromise between the Kabat CDRs andChothia structural loops, and are used by Oxford Molecular's AbMantibody modeling software. The “contact” hypervariable regions arebased on an analysis of the available complex crystal structures. Theresidues from each of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L24-L34 L30-L36 L2L50-L56 L50-L56 L50-L56 L46-L55 L3 L89-L97 L89-L97 L89-L97 L89-L96 H1H31-H35B H26-H35B H26-H32 . . . 34 H30-H35B (Kabat Numbering) H1 H31-H35H26-H35 H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H52-H56H47-H58 H3 H95-H102 H95-H102 H95-H102 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or95-102 (H3) in the VH. The variable domain residues are numberedaccording to Kabat et al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues or CDR residues herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), expressly incorporatedherein by reference). The “EU numbering system” or “EU index” isgenerally used when referring to a residue in an immunoglobulin heavychain constant region (e.g., the EU index reported in Kabat et al.,supra; hinge region in constant domain of heavy chain is approximatelyresidues 216-230 (EU numbering) of the heavy chain). The “EU index as inKabat” refers to the residue numbering of the human IgG1 EU antibody.Unless stated otherwise herein, references to residue numbers in thevariable domain of antibodies means residue numbering by the Kabatnumbering system. Unless stated otherwise herein, references to residuenumbers in the constant domain of antibodies means residue numbering bythe EU numbering system (e.g., see U.S. Provisional Application No.60/640,323, Figures for EU numbering).

As used herein, “polypeptide” refers generally to peptides and proteinshaving more than about ten amino acids. In one example, the polypeptideis a mammalian protein, examples of which include Factor D and fragmentsand/or variants of Factor D. In another example, the polypeptide is afull length antibody, or antibody fragment thereof (e.g. antigen-bindingfragment), that binds human Factor D, examples of which include Fab,Fab′, F(ab′)₂, and F_(v) fragments; diabodies, linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments (e.g. antigen-binding fragment).

A “variant” or “amino acid sequence variant” of a starting polypeptideis a polypeptide that comprises an amino acid sequence different fromthat of the starting polypeptide. Generally, a variant will possess atleast 80% sequence identity, preferably at least 90% sequence identity,more preferably at least 95% sequence identity, and most preferably atleast 98% sequence identity with the native polypeptide. Percentagesequence identity is determined for example, by the Fitch et al., Proc.Natl. Acad. Sci. USA, 80: 1382-1386 (1983), version of the algorithmdescribed by Needleman et al., J. Mol. Biol., 48: 443-453 (1970), afteraligning the sequences to provide for maximum homology. Amino acidsequence variants of a polypeptide may be prepared by introducingappropriate nucleotide changes into DNA encoding the polypeptide, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequence of the polypeptide of interest. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the polypeptide, such as changing thenumber or position of glycosylation sites. Methods for generating aminoacid sequence variants of polypeptides are described in U.S. Pat. No.5,534,615, expressly incorporated herein by reference, for example.

An “antibody variant” or “modified antibody” of a starting antibody isan antibody that comprises an amino acid sequence different from that ofthe starting antibody, wherein one or more of the amino acid residues ofthe starting antibody have been modified. Generally, an antibody variantwill possess at least 80% sequence identity, preferably at least 90%sequence identity, more preferably at least 95% sequence identity, andmost preferably at least 98% sequence identity with the startingantibody. Percentage sequence identity is determined for example, by theFitch et al., Proc. Natl. Acad. Sci. USA, 80: 1382-1386 (1983), versionof the algorithm described by Needleman et al., J. Mol. Biol., 48:443-453 (1970), after aligning the sequences of the starting antibodyand the candidate antibody variant to provide for maximum homology.Identity or similarity with respect to the parent sequenced is definedherein as the percentage of amino acid residues in the candidate variantsequence that are identical (i.e. same residue) or similar (i.e. aminoacid residue from the same group based on common side-chain properties,see below) with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Amino acid sequence variants of an antibodymay be prepared by introducing appropriate nucleotide changes into DNAencoding the antibody, or by peptide synthesis. Such variants include,for example, deletions from, and/or insertions into and/or substitutionsof, residues within the amino acid sequence of the antibody of interest.Any combination of deletion, insertion, and substitution is made toarrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid changes also mayalter post-translational processes of the antibody, such as changing thenumber or position of glycosylation sites. Methods for generatingantibody sequence variants of antibodies are similar to those forgenerating amino acid sequence variants of polypeptides described inU.S. Pat. No. 5,534,615, expressly incorporated herein by reference, forexample.

A “deamidated” variant of a polypeptide molecule is a polypeptidewherein one or more asparagine (N or Asn) residue(s) of the originalpolypeptide have been converted to aspartate (D or Asp), i.e. theneutral amide side chain has been converted to a residue with an overallacidic character. Deamidation may be prevented by converting asparagines(N or Asn) to glutamine (Q or Gln) or alanine (A or Ala) or serine (S orSer) (Amphlett, G. et al., Pharm. Biotechnol., 9:1-140 (1996)).

An “oxidized” variant of a polypeptide molecule is a polypeptide whereinone or more methionine (M or Met) or tryptophan (W or Trp) residue(s) ofthe original polypeptide have been converted to sulfone or sulfoxidethrough the sulfur of methionine. Oxidation may be prevented byconverting methionine (M or Met) to leucine (L or Leu) or isoleucine (Ior lie) (Amphlett, G. et al., Pharm. Biotechnol., 9:1-140 (1996)).

A “pyroglutamate” variant of a polypeptide molecule is a polypeptidewherein one or more glutamine (Q or Gln) residues(s) of the originalpolypeptide have been converted to pyroglutamate which occurs whenglutamine residues, for example N-terminal glutamine residues,spontaneously cyclize resulting in pyroglutamate. Pyroglutamateconversion may be prevented by converting glutamine (Q or Gln)residue(s) to glutamate (E or Glu) (Amphlett, G. et al., Pharm.Biotechnol., 9:1-140 (1996)).

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the target binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Thephrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-humanFactor D antibody is one which can bind to Factor D in such a manner soas to prevent or substantially reduce the complement activation. As usedherein, “functional fragment” with respect to antibodies, refers to Fv,F(ab) and F(ab′)₂ fragments. An “Fv” fragment is the minimum antibodyfragment which contains a complete target recognition and binding site.This region consists of a dimer of one heavy and one light chainvariable domain in a tight, non-covalent association (V_(H)-V_(L)dimer). It is in this configuration that the three CDRs of each variabledomain interact to define an target binding site on the surface of theV_(H)-V_(L) dimer. Collectively, the six CDRs confer target bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an target) has theability to recognize and bind target. “Single-chain Fv” or “sFv”antibody fragments comprise the V_(H) and V_(L) domains of an antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the sFv to form thedesired structure for target binding.

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments (e.g. antigen-binding fragments) are known to those ofordinary skill in the art.

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (for example, polypeptides of the invention), or thenucleic acids that encode these sequences, the sequences being differentin the combination of variant amino acids that are introduced into thesesequences according to the methods of the invention.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single targetic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on thetarget. In addition to their specificity, monoclonal antibodies areadvantageous in that they may be synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies for use with the presentinvention may be isolated from phage antibody libraries using the wellknown techniques. The parent monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256, 495 (1975),or may be made by recombinant methods.

“Humanized” forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibody mayalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin template chosen.

The terms “cell”, “cell line” and “cell culture” include progeny. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological property, as screened for inthe originally transformed cell, are included. The “host cells” used inthe present invention generally are prokaryotic or eukaryotic hosts.

The term “vector” means a DNA construct containing a DNA sequence whichis operably linked to a suitable control sequence capable of effectingthe expression of the DNA in a suitable host. Such control sequencesinclude a promoter to effect transcription, an optional operatorsequence to control such transcription, a sequence encoding suitablemRNA ribosome binding sites, and sequences which control the terminationof transcription and translation. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may in some instances, integrate into the genomeitself. In the present specification, “plasmid” and “vector” aresometimes used interchangeably, as the plasmid is the most commonly usedform of vector. However, the invention is intended to include such otherforms of vectors which serve equivalent function as and which are, orbecome, known in the art.

The word “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to a moleculeor protein, e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

As used herein, “solid phase” means a non-aqueous matrix to which theantibody of the present invention can adhere. Example of solid phasesencompassed herein include those formed partially or entirely of glass(e.g. controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column).

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target antigen with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman (1992) Curr. Opin.Struct. Biol. 3:355-362, and references cited therein. In a monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells (1991) Methods: A companion to Methodsin Enzymology 3:205-0216.

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of another Fc region by virtue of at least one “amino acidmodification” as herein defined. Preferably, the variant Fc region hasat least one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g. from about oneto about ten amino acid substitutions, and preferably from about one toabout five amino acid substitutions in a native sequence Fc region or inthe Fc region of the parent polypeptide. The variant Fc region hereinwill preferably possess at least about 80% homology with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% homology therewith, more preferablyat least about 95% homology therewith. Examples of “native sequencehuman Fc regions” are shown in FIG. 23 of WO 00/42072 and include anative sequence human IgG1 Fc region (non-A and A allotypes); nativesequence human IgG2 Fc region; native sequence human IgG3 Fc region; andnative sequence human IgG4 Fc region as well as naturally occurringvariants thereof. Native sequence murine Fc regions are shown in FIG.22A of WO 00/42072.

According to this invention, “altered” FcRn binding affinity is onewhich has either enhanced or diminished FcRn binding activity comparedto a parent polypeptide or to a polypeptide comprising a native sequenceFc region. In one example, the antibody with altered FcRn bindingaffinity has increased binding to FcRn at pH 6.0 and/or decreasedbinding to FcRn at pH 7.0. The variant which “displays increasedbinding” to an FcR binds at least one FcR with better affinity that theparent polypeptide. The variant which “displays decreased binding” to anFcR, binds at least one FcR with worse affinity than a parentpolypeptide. The variant which binds an FcR with “better affinity” thana parent polypeptide, is one which binds an FcR with substantiallybetter binding affinity than the parent antibody, when the amounts ofpolypeptide variant and parent polypeptide in the binding assay areessentially the same. For example, the polypeptide variant with improvedFcR binding affinity may display from about 1.15 fold to about 100 fold,e.g from about 1.2 fold to about 50 fold improvement in FcR bindingaffinity compared to the parent polypeptide, where FcR binding affinityis determined.

An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitution, insertion and/or deletion. Oneexample of an amino acid modification herein is a substitution.

An “amino acid modification at” a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specifiedresidues, or the insertion of at least one amino acid residues adjacentthe specified residue. By insertion “adjacent” a specified residue ismeant insertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e., encoded by the genetic code) and selected from the groupconsisting of: alanine (ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly), histidine (His); isoleucine (Ile); leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Substitution with one or more non-naturally occurring amino acidresidues is also encompassed by the definition of an amino acidsubstitution herein. A “non-naturally occurring amino acid residue”refers to a residue, other than those naturally occurring amino acidresidues listed above, which is able to covalently bind adjacent aminoacid residue(s) in a polypeptide chain. Examples of non-naturallyoccurring amino acid residues include norleucine, ornithine, norvaline,homoserine and other amino acid residue analogues such as thosedescribed in Ellman et al., Meth. Enzym, 202: 301-336 (1991). Togenerate such non-naturally occurring amino acid residues, theprocedures of Noren et al., Science, 244: 182 (1989) and Ellman et al.,supra, can be used. Briefly, these procedures involve chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence. While the insertionwill usually consist of the insertion of one or two amino acid residues,the present application contemplates larger “peptide insertions”, e.g.insertion of about three to about five or even up to about ten aminoacid residues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

II. Detailed Description

The invention herein provides Factor D antagonists, includinganti-Factor D antibodies, and variants thereof, and fragments thereof(e.g. antigen-binding fragments) useful for the prevention and treatmentof complement-associated conditions, including eye conditions (all eyeconditions and diseases the pathology of which involves complement,including the classical and the alternative pathways, and in particularthe alternative pathway of complement), such as, for example, maculardegenerative diseases, such as all stages of age-related maculardegeneration (AMD), including dry and wet (non-exudative and exudative)forms, choroidal neovascularization (CNV), uveitis, diabetic and otherischemia-related retinopathies, endophthalmitis, and other intraocularneovascular diseases, such as diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, and retinalneovascularization. One group of complement-associated eye conditionsincludes age-related macular degeneration (AMD), including non-exudative(e.g. intermediate dry AMD or geographic atrophy (GA)) and exudative(e.g. wet AMD (choroidal neovascularization (CNV)) AMD, diabeticretinopathy (DR), endophthalmitis and uveitis. In one example,complement-associated eye condition is intermediate dry AMD. In oneexample, complement-associated eye condition is geographic atrophy. Inone example, complement-associated eye condition is wet AMD (choroidalneovascularization (CNV)).

AMD is age-related degeneration of the macula, which is the leadingcause of irreversible visual dysfunction in individuals over the age of60. Two types of AMD exist, non-exudative (dry) and exudative (wet) AMD.The dry, or nonexudative, form involves atrophic and hypertrophicchanges in the retinal pigment epithelium (RPE) underlying the centralretina (macula) as well as deposits (drusen) on the RPE. Patients withnonexudative AMD can progress to the wet, or exudative, form of AMD, inwhich abnormal blood vessels called choroidal neovascular membranes(CNVMs) develop under the retina, leak fluid and blood, and ultimatelycause a blinding disciform scar in and under the retina. NonexudativeAMD, which is usually a precursor of exudative AMD, is more common. Thepresentation of nonexudative AMD varies: hard drusen, soft drusen, RPEgeographic atrophy, and pigment clumping can be present. Complementcomponents are deposited on the RPE early in AMD and are majorconstituents of drusen.

1. Humanized Anti-Factor D Antibodies

The invention herein includes the production and use of humanizedanti-Factor D antibodies, and fragments thereof. Exemplary methods forgenerating antibodies are described in more detail in the followingsections.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can in some instances be important toreduce antigenicity and/or HAMA response (human anti-mouse antibody)when the antibody is intended for human therapeutic use. Reduction orelimination of a HAMA response is generally a significant aspect ofclinical development of suitable therapeutic agents. See, e.g.,Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al.,Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985),135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller etal., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352;Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res.(1990), 50:1495. As described herein, the invention provides antibodiesthat are humanized such that HAMA response is reduced or eliminated.Variants of these antibodies can further be obtained using routinemethods known in the art, some of which are further described below.According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human V domainsequence which is closest to that of the rodent is identified and thehuman framework region (FR) within it accepted for the humanizedantibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J.Mol. Biol., 196:901 (1987)). Another method uses a particular frameworkregion derived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chains. The same framework may beused for several different humanized antibodies (Carter et al., Proc.Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993)).

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or hypervariable sequence(s). A selected frameworksequence to which a starting hypervariable sequence is linked isreferred to herein as an acceptor human framework. While the acceptorhuman frameworks may be from, or derived from, a human immunoglobulin(the VL and/or VH regions thereof), the acceptor human frameworks may befrom, or derived from, a human consensus framework sequence as suchframeworks have been demonstrated to have minimal, or no, immunogenicityin human patients. An “acceptor human framework” for the purposes hereinis a framework comprising the amino acid sequence of a VL or VHframework derived from a human immunoglobulin framework, or from a humanconsensus framework. An acceptor human framework “derived from” a humanimmunoglobulin framework or human consensus framework may comprise thesame amino acid sequence thereof, or may contain pre-existing amino acidsequence changes. Where pre-existing amino acid changes are present,preferably no more than 5 and preferably 4 or less, or 3 or less,pre-existing amino acid changes are present. In one embodiment, the VHacceptor human framework is identical in sequence to the VH humanimmunoglobulin framework sequence or human consensus framework sequence.In one embodiment, the VL acceptor human framework is identical insequence to the VL human immunoglobulin framework sequence or humanconsensus framework sequence. A “human consensus framework” is aframework which represents the most commonly occurring amino acidresidue in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal. In one embodiment, for the VH, the subgroup is subgroup III as inKabat et al.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence that is selected based onits homology to the donor framework sequence by aligning the donorframework sequence with various human framework sequences in acollection of human framework sequences, and select the most homologousframework sequence as the acceptor. The acceptor human framework may befrom or derived from human antibody germlne sequences available in thepublic databases.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup VII and/or VL kappa subgroup I consensusframework sequences.

In one embodiment, the human framework template used for generation ofan anti-Factor D antibody may comprise framework sequences from atemplate comprising a combination of VI-4.1b+ (VH7 family) and JH4d forVH chain and/or a combination of DPK4 (Vκl family) and JK2 for VL chain.

In one embodiment, the VH acceptor human framework comprises one, two,three or all of the following framework sequences:

(amino acids 1-25 of SEQ ID NO: 2) FR1 comprisingQVQLVQSGPELKKPGASVKVSCKAS, (amino acids 36-49 of SEQ ID NO: 2) FR2comprising WVRQAPGQGLE, (amino acids 67-98 of SEQ ID NO: 2) FR3comprising RFVFSLDTSVSTAYLQISSLKAEDTAVYYCER, (amino acids 67-97 of SEQID NO: 2) RFVFSLDTSVSTAYLQISSLKAEDTAVYYCE, (amino acids 67-96 of SEQ IDNO: 2) RFVFSLDTSVSTAYLQISSLKAEDTAVYYC, (SEQ ID NO: 3)RFVFSLDTSVSTAYLQISSLKAEDTAVYYCS, or (SEQ ID NO: 4)RFVFSLDTSVSTAYLQISSLKAEDTAVYYCSR, (amino acids 105-115 of SEQ ID NO: 2)FR4 comprising WGQGTLVTVSS.

In one embodiment, the VL acceptor human framework may comprise one,two, three or all of the following framework sequences:

(amino acids 1-23 of SEQ ID NO: 1) FR1 comprisingDIQVTQSPSSLSASVGDRVTITC, (amino acids 35-49 of SEQ ID NO: 1) FR2comprising WYQQKPGKVPKLLIS, (amino acids 57-88 of SEQ ID NO: 1) FR3comprising GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC, (amino acids 98-107 of SEQID NO: 1) FR4 comprising FGQGTKLEIK, or (SEQ ID NO: 5) FGQGTKVEIK.

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that be from a human immunoglobulin or ahuman consensus framework, the present invention contemplates that theacceptor sequence may comprise pre-existing amino acid substitutionsrelative to the human immunoglobulin sequence or human consensusframework sequence. These pre-existing substitutions are preferablyminimal; usually four, three, two or one amino acid differences onlyrelative to the human immunoglobulin sequence or consensus frameworksequence.

Hypervariable region residues of the non-human antibody are incorporatedinto the VL and/or VH acceptor human frameworks. For example, one mayincorporate residues corresponding to the Kabat CDR residues, theChothia hypervariable loop residues, the Abm residues, and/or contactresidues. Optionally, the extended hypervariable region residues asfollows are incorporated: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and89-97 (L3), 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102,or 95-102 (H3).

In one aspect, the invention provides an anti-Factor D antibodycomprising a heavy chain variable domain comprising SEQ ID NO: 2. In oneaspect, the invention provides an anti-Factor D antibody comprising alight chain variable domain comprising SEQ ID NO: 1. In one aspect, theinvention provides an anti-Factor D antibody comprising a heavy chainvariable domain comprising SEQ ID NO: 2 and a light chain variabledomain comprising SEQ ID NO: 1. In one example, the invention provides afragment of said anti-Factor D antibodies (e.g. antigen-bindingfragments).

In one aspect, the invention provides an anti-Factor D antibodycomprising a heavy chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to an amino acid sequence of SEQ ID NO: 2. In someembodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity contains substitutions,insertions, or deletions relative to the reference sequence, but anantibody comprising that amino acid sequence retains the ability to bindto Factor D. In some embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted, or deleted in a sequence of SEQ ID NO: 2. Insome embodiments, the substitutions, insertions or deletions occur inregions outside the HVRs (i.e., in the FRs). In some embodiments, ananti-Factor D antibody comprises a heavy chain variable domaincomprising an amino acid sequence of SEQ ID NO: 2. In one example, theinvention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

In some embodiments, the invention provides an anti-Factor D antibodycomprising a light chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to an amino acid sequence of SEQ ID NO: 1. In someembodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity contains substitutions,insertions, or deletions relative to the reference sequence, but anantibody comprising that amino acid sequence retains the ability to bindto Factor D. In some embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted, or deleted in a sequence of SEQ ID NO: 1. Insome embodiments, the substitutions, insertions or deletions occur inregions outside the HVRs (i.e., in the FRs). In some embodiments, ananti-Factor D antibody comprises a light chain variable domaincomprising an amino acid sequence of SEQ ID NO: 1. In one example, theinvention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

An anti-Factor D antibody may comprise any suitable framework variabledomain sequence, provided that the antibody retains the ability to bindFactor D. For example, in some embodiments, anti-Factor D antibodies ofthe invention comprise a heavy chain variable domain framework sequencethat is a combination of VI.4.1 b+ and JH4d (See FIG. 3). In someembodiments, anti-Factor D antibodies of the invention comprise a humansubgroup VII heavy chain framework consensus sequence. In someembodiments, anti-Factor D antibodies of the invention comprise a heavychain variable domain framework sequence comprising FR1 comprising aminoacids 1-25 of SEQ ID NO: 2, FR2 comprising amino acids 36-49 of SEQ IDNO: 2, FR3 comprising amino acids 67-98 of SEQ ID NO: 2 and FR4comprising amino acids 105-115 of SEQ ID NO: 2 In one embodiment ofthese antibodies, the heavy chain variable domain sequence comprisessubstitution(s) at position 40 and/or 88 (Kabat numbering). In oneembodiment of these antibodies, position 40 is cysteine (C) or alanine(A) and/or position 88 is cysteine (C) or alanine (A). In someembodiments, anti-Factor D antibodies of the invention comprise a lightchain variable domain framework sequence that is a combination of DPK4and JK2 (See FIG. 4). In some embodiments, anti-Factor D antibodies ofthe invention comprise a human kappa I (κI) light chain frameworkconsensus sequence. In some embodiments, anti-Factor D antibodies of theinvention comprise a light chain variable domain framework sequencecomprising FR1 comprising amino acids 1-23 of SEQ ID NO: 1, FR2comprising amino acids 35-49 of SEQ ID NO: 1, FR3 comprising amino acids57-88 of SEQ ID NO: 1 and FR4 comprising amino acids 98-107 of SEQ IDNO: 1. In one embodiment of these antibodies, the light chain variableframework sequence comprises one or more substitution(s) at position 15,43 and/or 104, (Kabat numbering). In one embodiment of these antibodies,position 15 is cysteine (C) or valine (V), position 43 is cysteine (C)or alanine (A), position 104 is valine (V) or leucine (L). In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

Further, an anti-Factor D antibody may comprise any suitable constantdomain sequence, provided that the antibody retains the ability to bindFactor D. For example, in some embodiments, anti-Factor D antibodies ofthe invention comprise at least a portion of a heavy chain constantdomain. In one embodiment, anti-Factor D antibodies of the inventioncomprise a heavy chain constant domain of either one or a combination ofan α, δ, ε, γ, or μ heavy chain. Depending on the amino acid sequence ofthe constant domain of their heavy chains (C_(H)), immunoglobulins canbe assigned to different classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chainsdesignated α, δ, ε, γ, and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in C_(H) sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In oneembodiment, anti-Factor D antibodies of the invention comprise a heavychain constant domain comprising substitutions at amino acid positionsthat results in a desired effect on effector function (e.g. bindingaffinity). In one embodiment, anti-Factor D antibodies of the inventioncomprise a heavy chain constant domain comprising substitutions at aminoacid positions that do not result in an effect on effector function(e.g. binding affinity). In one embodiment, anti-Factor D antibodies ofthe invention comprise a heavy chain constant domain of the IgG type(e.g. IgG1, IgG2, IgG3 or IgG4) and further comprise a substitution atposition 114 (Kabat numbering; equivalent to 118 in EU numbering), 168(Kabat numbering; equivalent to 172 in EU numbering), 172 (Kabatnumbering; equivalent to 176 in EU numbering) and/or 228 (EU numbering).In one embodiment, anti-Factor D antibodies of the invention comprise aheavy chain constant domain of the IgG (e.g. IgG1, IgG2, IgG3 or IgG4)type and further comprise a substitution at position 114 whereinposition 114 is a cysteine (C) or alanine (A), position 168 is cysteine(C) or alanine (A), position 172 is a cysteine (C) or alanine (A) and/orposition 228 is a proline (P), arginine (R) or serine (S). In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

Further, for example, in some embodiments, anti-Factor D antibodies ofthe invention comprise at least a portion of a light chain constantdomain. In one embodiment, anti-Factor D antibodies of the inventioncomprise a light chain constant domain of either one or a combination ofa kappa or a lambda light chain, as the light chain from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. In one embodiment, anti-Factor D antibodies of the inventioncomprise a light chain constant domain comprising substitutions at aminoacid positions that results in a desired effect on effector function(e.g. binding affinity). In one embodiment, anti-Factor D antibodies ofthe invention comprise a light chain constant domain comprisingsubstitutions at amino acid positions that do not result in an effect oneffector function (e.g., binding affinity). In one embodiment,anti-Factor D antibodies of the invention comprise a light chainconstant domain of the kappa type and further comprise a substitution atposition 110, 144, 146 and/or 168 (Kabat numbering). In one embodiment,anti-Factor D antibodies of the invention comprise a light chainconstant domain of the kappa type and further comprise a substitution atposition 110 wherein 110 is a cysteine (C) or valine (V), at position144 wherein 144 is a cysteine (C) or alanine (A), at position 146wherein 146 is a isoleucine (I) or valine (V) and/or at position 168wherein 168 is a cysteine (C) or serine (S). In one example, theinvention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

2. Modified Anti-Factor D Antibodies

The invention herein includes the production and use of modifiedanti-Factor D antibodies, for example modified humanized anti-Factor Dantibodies, and variants thereof, and fragments thereof (e.g.antigen-binding fragments). Exemplary methods for generating modifiedantibodies are described in more detail in the following sections.

A parent anti-Factor D antibody, including a humanized anti-Factor Dantibody, can be modified to generate modified anti-Factor D antibodies,and variants thereof. In one embodiment, the modified anti-Factor Dantibodies, and variants thereof, may have improved physical, chemical,biological or homogeneity properties over the parent antibody. In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In one embodiment, an antibody of this invention comprises one or moreamino acid alterations (e.g. substitutions) into one or more of thehypervariable regions of the parent antibody. Alternatively, or inaddition, one or more alterations (e.g. substitutions) of frameworkregion residues may be introduced in the parent antibody. Examples offramework region residues to modify include those which non-covalentlybind antigen directly (Amit et al., (1986) Science, 233: 747-753);interact with/effect the conformation of a CDR (Chothia et al. (1987) J.Mol. Biol., 196: 901-917), and/or participate in the V_(L)-V_(H)interface (EP 239 400B1). In certain embodiments, modification of one ormore of such framework region residues results in an enhancement of thebinding affinity of the antibody for the antigen. For example, fromabout one to about 5 framework residues may be altered in thisembodiment of the invention. Examples of framework or HVR regionresidues to modify include sites, wherein modifications at such sitesresult in the generation of deamidated variants (for example, asparagine(N or Asn) residue(s) modified to aspartate (D or Asp), oxidationvariants (for example, methionine (M or Met) residue(s) and/ortryptophan (W or Trp) residue(s) modified to sulfone or sulfoxide) orpyroglutamate variants (for example, glutamine (Q or Gln) residue(s)modified to pyroglutamate). Examples of framework region residues or HVRregion residues to modify include possible deamidation sites (i.e.asparagine (N or Asn)), oxidation sites (i.e. methionine (M or Met) ortryptophan (W or Trp)) or pyroglutamate conversion sites (i.e. glutamine(Q or Gln)), wherein modification at such sites prevent deamidationand/or oxidation and/or pyroglutamate conversion, respectively. Toprevent the formation of deamidated variants, asparagine (N or Asn) maybe mutated to alanine (A or Ala), glutamine (Q or Gln) or serine (S orSer). To prevent the formation of oxidated variants, methionine (Met) ortryptophan (W or Trp) may be mutated to leucine (L) or isoleucine (I).To prevent the formation of pyroglutamate variants, glutamine (Q or Gln)may be mutated to glutamate (E or Glu). (Amphlett, G. et al., Pharm.Biotechnol., 9:1-140 (1996)). Alternatively, or in addition, one or morealterations (e.g. substitutions) of framework region residues may be inthe Fc region in the parent antibody. In one example, the inventionprovides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

In one embodiment, an antibody of this invention comprises a variant Fcregion such that the half-life of the antibody in vivo is increased ordecreased relative to the parent antibody or the antibody comprising anative sequence Fc region. In one embodiment, the antibody comprises anvariant Fc region that increases or decreases neonatal Fc receptor(FcRn) binding affinity to the antibody (see WO2000042072, incorporatedby reference in its entirety). For example, such antibody can comprisean amino acid modification at any one or more of amino acid positions238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400,413, 415, 424, 433, 434, 435, 436, 439 or 447 of the Fc region, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat. Such polypeptide variants with reduced binding to an FcRnmay comprise an amino acid modification at any one or more of amino acidpositions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435,436, 439 or 447 of the Fc region, wherein the numbering of the residuesin the Fc region is that of the EU index as in Kabat. Theabove-mentioned polypeptide variants may, alternatively, displayincreased binding to FcRn and comprise an amino acid modification at anyone or more of amino acid positions 238, 256, 265, 272, 286, 303, 305,307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or434 of the Fc region, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat. For example, the antibodycomprises a variant Fc region that binds with increased half-life invivo relative to the parent antibody or the antibody comprising a nativesequence Fc region. For example, the antibody comprises a variant Fcregion that binds with increased affinity to FcRn relative to the parentantibody or the antibody comprising a native sequence Fc region.

FcRn Binding Affinity May be Measured as Follows

The binding of antibodies of this invention against human FcRn can bestudied by surface plasmon resonance using, for example, a BiaCore 3000instrument. Human FcRn is coupled to a sensor chip using an aminecoupling kit. For example, a CM5 sensor chip can be activated withEDC/NHS for 7 min at 5 μl/min. 100 μg/ml of human FcRn can be injectedfor 30 sec to 2 min at a flow rate of 10 μl/min over the activated chipto give a final binding response unit (RU) of 100 to 200. Afterconjugation, the FcRn coupled chip can be blocked by an injection of 35μl of 1M ethanolamine hydrochloride at 5 μl/min.

The binding of the antibodies of this invention to human FcRn at pH 6.0or pH 7.4 can be determined. The running buffer for the bindingexperiment is either PBS pH 6.0 or pH 7.4 containing 0.01% P20 and 0.02%sodium azide. Antibodies of this invention can be buffer-exchanged intoeither pH 6.0 or pH 7.4 running buffer. In one embodiment, theexperiments are performed at 25° C. For the pH 6.0 run, antibodies, withconcentrations ranging from 4 μM to 0.7 nM, are flowed over an FcRncoated chip at 30 μl/min for 4 min and then are allowed to dissociatefrom the chip for 5 min. For the pH 7.4 run, antibodies, withconcentrations ranging from 12 μM to 100 nM, are injected over the FcRncoated chip at 20 μl/min for 1.5 min and then released for 2 min.Antibodies are also flowed over an unconjugated spot on the sensor chipto allow subtraction of background non-specific binding from the bindingto FcRn-coupled chip. Chip can be regenerated with 30 sec pulse of 0.1MTRIS pH 8.3 in between injections. Steady state RU for each injectioncan be recorded at the end of each injection phase, and dissociationconstants (K_(D)) are later calculated by plotting the steady state RUagainst injection concentration.

One useful procedure for generating such modified antibodies, andfragments thereof (e.g. antigen-binding fragments) and variants thereof,is called “alanine scanning mutagenesis” (Cunningham and Wells (1989)Science 244:1081-1085). Here, one or more of the hypervariable regionresidue(s) are replaced by alanine or polyalanine residue(s) to affectthe interaction of the amino acids with the antigen. Those hypervariableregion residue(s) demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other mutationsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. The ala-mutantsproduced this way are screened for their biological activity (i.e.binding affinity or hemolysis assay) as described herein.

Normally one would start with a conservative substitution such as thoseshown below under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity (e.g. bindingaffinity), then more substantial changes, denominated “exemplarysubstitutions” in the following table, or as further described below inreference to amino acid classes, are introduced and the productsscreened. Preferred substitutions are listed in the table below.

TABLE 1 Preferred Substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; lys; arg gln Asp (D) Glu glu Cys (C) Ser serGln (Q) Asn asn Glu (E) Asp asp Gly (G) pro; ala ala His (H) asn; gln;lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu (L)norleucine; ile; val; met; ala; ile phe Lys (K) arg; gln; asn arg Met(M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) Alaala Ser (S) Thr thr Thr (T) Ser ser Trp (W) tyr; phe tyr Tyr (Y) trp;phe; thr; ser phe Val (V) ile; leu; met; phe; ala; leu norleucine

Even more substantial modifications in the antibodies or fragmentsthereof (e.g. antigen-binding fragments) biological properties areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr, asn, gln;    -   (3) acidic: asp, glu;    -   (4) basic: his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

In another embodiment, the sites selected for modification are modified,and those modifications with improved binding affinity are selected byphage display.

Nucleic acid molecules encoding amino acid sequence mutants or modifiedamino acid sequences are prepared by a variety of methods known in theart. These methods include, but are not limited to,oligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the parent antibody. One method for makingmutants or variants or modified amino acid sequences is site directedmutagenesis (see, e.g., Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488).

In certain embodiments, the modified antibody will only have a singlehypervariable region residue substituted. In other embodiments, two ormore of the hypervariable region residues of the parent antibody willhave been substituted, e.g. from about two to about ten hypervariableregion substitutions. In one example, the invention provides a fragmentof said anti-Factor D antibodies (e.g. antigen-binding fragments).

Ordinarily, the modified antibody will have an amino acid sequencehaving at least 75% amino acid sequence identity or similarity (definedabove in Definition section) with the amino acid sequence of either theheavy or light chain variable domain of the parent antibody, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, and most preferably at least 95%. In one example, theinvention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

Following production of the modified antibody, or fragment thereof (e.g.antigen-binding fragment) the biological activity of that moleculerelative to the parent antibody, or fragment thereof (e.g.antigen-binding fragment) is determined. As noted above, this mayinvolve determining the binding affinity and/or other biologicalactivities of the antibody variant, or fragment thereof (e.g.antigen-binding fragment). In one embodiment of the invention, a panelof modified antibodies, or fragments thereof (e.g. antigen-bindingfragments) is prepared and screened for binding affinity for the antigensuch as Factor D or a fragment thereof. One or more of the antibodymutants or modified antibodies, or fragments thereof (e.g.antigen-binding fragments) selected from this initial screen areoptionally subjected to one or more further biological activity assaysto confirm that the antibody variant(s), or fragments thereof (e.g.antigen-binding fragments) are indeed useful, e.g. for preclinicalstudies.

The modified anti-Factor D antibodies, or fragments thereof (e.g.antigen-binding fragments) described herein may be subjected to furthermodifications, oftentimes depending on the intended use of the modifiedantibody, or fragment thereof (e.g. antigen-binding fragment). Suchmodifications may involve further alteration of the amino acid sequence,fusion to heterologous polypeptide(s) and/or covalent modifications suchas those elaborated below. With respect to amino acid sequencealterations, exemplary modifications are elaborated above. For example,any cysteine residue not involved in maintaining the proper conformationof the modified antibody also may be substituted, generally with serine,to improve the oxidative stability of the molecule and prevent aberrantcross linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment). Another type of amino acid mutant hasan altered glycosylation pattern. This may be achieved by deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies, or antibody fragments (e.g. antigen-bindingfragments) is typically either N-linked or O-linked. N-linked refers tothe attachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone of the sugars N-aceylgalactosamine, galactose, or xylose to ahydroxyamino acid, most commonly serine or threonine, although5-hydroxyproline or 5-hydroxylysine may also be used. Addition ofglycosylation sites to the antibody is conveniently accomplished byaltering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

In one embodiment, the invention provides a modified anti-Factor Dantibody, of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leastone position (according to Kabat numbering) of the amino acid sequenceof such parent anti-Factor D antibody of the application is substitutedwith one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody variant comprises the amino acidsequence of such parent anti-Factor D antibody of the application,wherein at least one position (according to Kabat numbering) of theamino acid sequence of such parent anti-Factor D antibody of theapplication is substituted with one or more of the following:

-   -   (a) amino acid at position 1 of the heavy chain is E;    -   (b) amino acid at position 99 of the heavy chain is A or Q; or    -   (c) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leastone position (according to Kabat numbering) of the amino acid sequenceof such parent anti-Factor D antibody of the application is substitutedwith one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V;    -   (e) amino acid at position 1 of the heavy chain is E;    -   (f) amino acid at position 99 of the heavy chain is A or Q; or    -   (g) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leasttwo positions (according to Kabat numbering) of the amino acid sequenceof such parent anti-Factor D antibody of the application is substitutedwith one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V.

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leasttwo positions (according to Kabat numbering) of the amino acid sequenceof such parent anti-Factor D antibody of the application is substitutedwith one or more of the following:

-   -   (a) amino acid at position 1 of the heavy chain is E;    -   (b) amino acid at position 99 of the heavy chain is A or Q; or    -   (c) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leasttwo positions (according to Kabat numbering) of the amino acid sequenceof such parent anti-Factor D antibody of the application is substitutedwith one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V;    -   (e) amino acid at position 1 of the heavy chain is E;    -   (f) amino acid at position 99 of the heavy chain is A or Q; or    -   (g) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leastthree positions (according to Kabat numbering) of the amino acidsequence of such parent anti-Factor D antibody of the application issubstituted with one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leastthree positions (according to Kabat numbering) of the amino acidsequence of such parent anti-Factor D antibody of the application issubstituted with one or more of the following:

-   -   (a) amino acid at position 1 of the heavy chain is E;    -   (b) amino acid at position 99 of the heavy chain is A or Q; or    -   (c) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody of a parent anti-Factor D antibody of the application, whereinthe modified anti-Factor D antibody comprises the amino acid sequence ofsuch parent anti-Factor D antibody of the application, wherein at leastthree positions (according to Kabat numbering) of the amino acidsequence of such parent anti-Factor D antibody of the application issubstituted with one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V;    -   (e) amino acid at position 1 of the heavy chain is E;    -   (f) amino acid at position 99 of the heavy chain is A or Q; or    -   (g) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides an anti-Factor D antibodycomprising light chain HVRs of a reference antibody, wherein saidanti-Factor D antibody further comprises a substitution at one or morepositions of said reference antibody, wherein said reference antibodycomprises light chain HVR-1 comprising ITSTDIDDDMN (SEQ ID NO: 30),light chain HVR-2 comprising GGNTLRP (SEQ ID NO: 35), and light chainHVR-3 comprising LQSDSLPYT (SEQ ID NO: 38), and wherein saidsubstitution is one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V;    -   (e) amino acid at position 1 of the heavy chain is E;    -   (f) amino acid at position 99 of the heavy chain is A or Q; or    -   (g) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides an anti-Factor D antibodycomprising heavy chain HVRs of a reference antibody, wherein saidanti-Factor D antibody further comprises a substitution at one or morepositions of said reference antibody, wherein said reference antibodycomprises heavy chain HVR-1 comprising GYTFTNYGMN (SEQ ID NO: 39), heavychain HVR-2 comprising WINTYTGETTYADDFKG (SEQ ID NO: 40), and heavychain HVR-3 comprising EGGVNN (SEQ ID NO: 41), and wherein saidsubstitution is one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V′    -   (e) amino acid at position 1 of the heavy chain is E;    -   (f) amino acid at position 99 of the heavy chain is A or Q; or    -   (g) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides an anti-Factor D antibodycomprising light chain HVRs and heavy chain HVRs of a referenceantibody, wherein said anti-Factor D antibody further comprises asubstitution at one or more positions of said reference antibody,wherein said reference antibody comprises light chain HVR-1 comprisingITSTDIDDDMN (SEQ ID NO: 30), light chain HVR-2 comprising GGNTLRP (SEQID NO: 35), and light chain HVR-3 comprising LQSDSLPYT (SEQ ID NO: 38),and heavy chain HVR-1 comprising GYTFTNYGMN (SEQ ID NO: 39), heavy chainHVR-2 comprising WINTYTGETTYADDFKG (SEQ ID NO: 40), and heavy chainHVR-3 comprising EGGVNN (SEQ ID NO: 41), and wherein said substitutionis one or more of the following:

-   -   (a) amino acid at position 33 of the light chain is L or I;    -   (b) amino acid at position 34 of the light chain is A or Q;    -   (c) amino acid at position 52 of the light chain is S or A;    -   (d) amino acid at position 104 of the light chain is V;    -   (e) amino acid at position 1 of the heavy chain is E;    -   (f) amino acid at position 99 of the heavy chain is A or Q; or    -   (g) amino acid at position 100 of the heavy chain is A or Q.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one aspect, the invention provides a modified anti-Factor D antibodycomprising:

-   -   (a) at least one, two, three, four, five or six HVRs selected        from:        -   (i) an HVR-H1 comprising an amino acid sequence having at            least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%            sequence identity to an amino acid sequence selected from            SEQ ID NO: 39;        -   (ii) an HVR-H2 comprising an amino acid sequence having at            least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%            sequence identity to an amino acid sequence selected from            SEQ ID NO: 40;        -   (iii) an HVR-H3 comprising an amino acid sequence having at            least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%            sequence identity to an amino acid sequence selected from            SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44            and SEQ ID NO: 45;        -   (iv) an HVR-L1 comprising an amino acid sequence having at            least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%            sequence identity to an amino acid sequence selected from            SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33            and SEQ ID NO: 34;        -   (v) an HVR-L2 comprising an amino acid sequence having at            least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%            sequence identity to an amino acid sequence selected from            SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37; and        -   (vi) an HVR-L3 comprising an amino acid sequence having at            least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%            sequence identity to an amino acid sequence selected from            SEQ ID NO: 38; or    -   (b) at least one variant HVR, wherein the variant HVR comprises        modification of at least one residue of the sequence depicted in        SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 30, 31, 32, 33, 34, 35,        36, 37 or 38.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In some embodiments, an HVR having an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity contains substitutions, insertions, or deletions relative tothe reference sequence, but an antibody comprising that amino acidsequence retains the ability to bind to Factor D. In some embodiments, atotal of 1 to 10 amino acids have been substituted, inserted, or deletedin the reference sequence selected from the group consisting of SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37 and SEQ ID NO: 38.

In one aspect, the invention provides a modified anti-Factor D antibodycomprising:

-   -   (a) at least one, two, three, four, five or six HVRs selected        from:        -   (i) an HVR-H1 comprising the amino acid sequence selected            from SEQ ID NO: 39;        -   (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID            NO: 40;        -   (iii) an HVR-H3 comprising the amino acid sequence selected            from SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:            44 and SEQ ID NO: 45;        -   (iv) an HVR-L1 comprising the amino acid sequence selected            from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:            33 and SEQ ID NO: 34;        -   (v) an HVR-L2 comprising the amino acid sequence selected            from SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37; and        -   (vi) an HVR-L3 comprising the amino acid sequence selected            from SEQ ID NO: 38; or    -   (b) at least one variant HVR, wherein the variant HVR comprises        modification of at least one residue of the sequence depicted in        SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 30, 31, 32, 33, 34, 35,        36, 37 or 38.        In one example, the invention provides a fragment of said        anti-Factor D antibody (e.g. antigen-binding fragment).

In one embodiment, the invention provides a modified anti-Factor Dantibody comprising an HVR-H1 comprising the amino acid sequence of SEQID NO: 39. In another embodiment, the invention provides a modifiedanti-Factor D antibody comprising an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 40. In another embodiment, the invention providesa modified anti-Factor D antibody comprising an HVR-H3 comprising theamino acid sequence of SEQ ID NO: 41. In another embodiment, theinvention provides a modified anti-Factor D antibody comprising anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 30. In anotherembodiment, the invention provides a modified anti-Factor D antibodycomprising an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35. In another embodiment, the invention provides a modified anti-FactorD antibody comprising the amino acid sequence of SEQ ID NO: 38. In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In another embodiment, the invention provides a modified anti-Factor Dantibody comprising an HVR-H1 comprising the amino acid sequence of SEQID NO: 39, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:40, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41.In one example, the invention provides a fragment of said anti-Factor Dantibody (e.g. antigen-binding fragment).

In another embodiment, the invention provides a modified anti-Factor Dantibody comprising an HVR-L1 comprising the amino acid sequence of SEQID NO: 30, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.In one example, the invention provides a fragment of said anti-Factor Dantibody (e.g. antigen-binding fragment).

In another embodiment, the invention provides a modified anti-Factor Dantibody comprising an HVR-H1 comprising the amino acid sequence of SEQID NO: 39, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:40, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41, anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 30, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 35, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 38. In one example, theinvention provides a fragment of said anti-Factor D antibody (e.g.antigen-binding fragment).

In one aspect, the invention provides a modified anti-Factor D antibodycomprising a heavy chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and29. In some embodiments, an amino acid sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity containssubstitutions, insertions, or deletions relative to the referencesequence, but an antibody comprising that amino acid sequence retainsthe ability to bind to Factor D. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, or deleted in a sequenceselected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28 and 29. In some embodiments, the substitutions,insertions or deletions occur in regions outside the HVRs (i.e., in theFRs). In some embodiments, a modified anti-Factor D antibody comprises aheavy chain variable domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28 and 29. In one example, the invention provides a fragment ofsaid anti-Factor D antibodies (e.g. antigen-binding fragments).

In one aspect, the invention provides a modified anti-Factor D antibodycomprising a light chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.In some embodiments, an amino acid sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity containssubstitutions, insertions, or deletions relative to the referencesequence, but an antibody comprising that amino acid sequence retainsthe ability to bind to Factor D. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, or deleted in a sequenceselected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 and 17. In some embodiments, the substitutions,insertions or deletions occur in regions outside the HVRs (i.e., in theFRs). In some embodiments, a modified anti-Factor D antibody comprises alight chain variable domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 and 17. In one example, the invention provides a fragment of saidanti-Factor D antibodies (e.g. antigen-binding fragments).

For example, the invention provides a modified anti-Factor D antibodycomprising a heavy chain variable domain comprising SEQ ID NO: 18. Forexample, the invention provides a modified anti-Factor D antibodycomprising a light chain variable domain comprising SEQ ID NO: 6. Forexample, the invention provides a modified anti-Factor D antibodycomprising a heavy chain variable domain comprising SEQ ID NO: 18 and alight chain variable domain comprising SEQ ID NO: 6. For example, theinvention provides a modified anti-Factor D antibody comprising a heavychain variable domain comprising SEQ ID NO: 19. For example, theinvention provides a modified anti-Factor D antibody comprising a lightchain variable domain comprising SEQ ID NO: 7. For example, theinvention provides a modified anti-Factor D antibody comprising a heavychain variable domain comprising SEQ ID NO: 19 and a light chainvariable domain comprising SEQ ID NO: 7. For example, the inventionprovides a modified anti-Factor D antibody comprising a heavy chainvariable domain comprising SEQ ID NO: 20. For example, the inventionprovides a modified anti-Factor D antibody comprising a light chainvariable domain comprising SEQ ID NO: 8. For example, the inventionprovides a modified anti-Factor D antibody comprising a heavy chainvariable domain comprising SEQ ID NO: 20 and a light chain variabledomain comprising SEQ ID NO: 8. For example, the invention provides amodified anti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 21. For example, the invention provides a modifiedanti-Factor D antibody comprising a light chain variable domaincomprising SEQ ID NO: 9. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 21 and a light chain variable domain comprisingSEQ ID NO: 9. For example, the invention provides a modified anti-FactorD antibody comprising a heavy chain variable domain comprising SEQ IDNO: 22. For example, the invention provides a modified anti-Factor Dantibody comprising a light chain variable domain comprising SEQ ID NO:10. For example, the invention provides a modified anti-Factor Dantibody comprising a heavy chain variable domain comprising SEQ ID NO:22 and a light chain variable domain comprising SEQ ID NO: 10. Forexample, the invention provides a modified anti-Factor D antibodycomprising a heavy chain variable domain comprising SEQ ID NO: 23. Forexample, the invention provides a modified anti-Factor D antibodycomprising a light chain variable domain comprising SEQ ID NO: 11. Forexample, the invention provides a modified anti-Factor D antibodycomprising a heavy chain variable domain comprising SEQ ID NO: 23 and alight chain variable domain comprising SEQ ID NO: 11. For example, theinvention provides a modified anti-Factor D antibody comprising a heavychain variable domain comprising SEQ ID NO: 24. For example, theinvention provides a modified anti-Factor D antibody comprising a lightchain variable domain comprising SEQ ID NO: 12. For example, theinvention provides a modified anti-Factor D antibody comprising a heavychain variable domain comprising SEQ ID NO: 24 and a light chainvariable domain comprising SEQ ID NO: 12. For example, the inventionprovides a modified anti-Factor D antibody comprising a heavy chainvariable domain comprising SEQ ID NO: 25. For example, the inventionprovides a modified anti-Factor D antibody comprising a light chainvariable domain comprising SEQ ID NO: 13. For example, the inventionprovides a modified anti-Factor D antibody comprising a heavy chainvariable domain comprising SEQ ID NO: 25 and a light chain variabledomain comprising SEQ ID NO: 13. For example, the invention provides amodified anti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 26. For example, the invention provides a modifiedanti-Factor D antibody comprising a light chain variable domaincomprising SEQ ID NO: 14. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 26 and a light chain variable domain comprisingSEQ ID NO: 14. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 27. For example, the invention provides a modifiedanti-Factor D antibody comprising a light chain variable domaincomprising SEQ ID NO: 15. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 27 and a light chain variable domain comprisingSEQ ID NO: 15. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 28. For example, the invention provides a modifiedanti-Factor D antibody comprising a light chain variable domaincomprising SEQ ID NO: 16. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 28 and a light chain variable domain comprisingSEQ ID NO: 16. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 29. For example, the invention provides a modifiedanti-Factor D antibody comprising a light chain variable domaincomprising SEQ ID NO: 17. For example, the invention provides a modifiedanti-Factor D antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 29 and a light chain variable domain comprisingSEQ ID NO: 17. In one example, the invention provides a fragment of saidanti-Factor D antibodies (e.g. antigen-binding fragments).

In one embodiment, the invention provides a polypeptide comprising thefollowing amino acid sequence:X₁VQLVQSGPELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGETTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCEREGGVX₂X₃WGQGTLVTVSS (SEQ ID NO:74), wherein X₁ is Q or E; X₂ is N, A or Q; and X₃ is N, A or Q. In oneembodiment, the invention provides a modified anti-Factor D antibodycomprising the following amino acid sequence:X₁VQLVQSGPELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGETTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCEREGGVX₂X₃WGQGTLVTVSS (SEQ ID NO:74), wherein X₁ is Q or E; X₂ is N, A or Q; and X₃ is N, A or Q. In oneexample, the invention provides a fragment of said polypeptide.

In one embodiment, the invention provides a polypeptide comprisingfollowing amino acid sequence:DIQVTQSPSSLSASVGDRVTITCITSTDIDDDX₄X₅WYQQKPGKVPKLLISGGX₆TLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDSLPYTFGQGTKX₇EIK (SEQ ID NO: 73), whereinX₄ is M, L or I; X₅ is N, A or Q; X₆ is N, S or A; and X₇ is L or V. Inone embodiment, the invention provides a modified anti-Factor D antibodycomprising the polypeptide comprising the following amino acid sequence:DIQVTQSPSSLSASVGDRVTITCITSTDIDDDX₄X₅WYQQKPGKVPKLLISGGX₆TLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDSLPYTFGQGTKX₇EIK (SEQ ID NO: 73), whereinX₄ is M, L or I; X₅ is N, A or Q; X₆ is N, S or A; and X₇ is L or V. Inone example, the invention provides a fragment of said polypeptide.

In one embodiment, the invention provides a polypeptide comprising anamino acid sequence selected from the group comprising SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28 and SEQ ID NO: 29. In another embodiment, the invention providesa polypeptide comprising an amino acid sequence selected from the groupcomprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17. In one example, theinvention provides a fragment of said polypeptide.

In one aspect, the invention provides a modified anti-Factor D antibody,wherein the light chain domain comprises the sequence of SEQ ID NO: 47.In another aspect, the invention provides a modified anti-Factor Dantibody, wherein the heavy chain domain comprises the sequence of SEQID NO: 54. In one aspect, the invention provides a modified anti-FactorD antibody, wherein the light chain domain comprises the sequence of SEQID NO: 61. In another aspect, the invention provides a modifiedanti-Factor D antibody, wherein the heavy chain domain comprises thesequence of SEQ ID NO:63. In one example, the invention provides afragment of said anti-Factor D antibodies (e.g. antigen-bindingfragments).

In one aspect, the invention provides a polypeptide comprising thesequence of SEQ ID NO: 47. In another aspect, the invention provides apolypeptide comprising the sequence of SEQ ID NO: 54. In one aspect, theinvention provides a polypeptide comprising the sequence of SEQ ID NO:61. In another aspect, the invention provides a polypeptide comprisingthe sequence of SEQ ID NO: 63. In one example, the invention provides afragment of said polypeptide.

In one aspect, the invention provides a modified anti-Factor D antibodyof humanized anti-Factor D #111, wherein the variable light chain domaincomprises the amino acid sequence selected from the group consisting ofSEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16 and SEQ ID NO: 17. In one example, the inventionprovides a fragment of said anti-Factor D antibody (e.g. antigen-bindingfragment).

In one aspect, the invention provides a modified anti-Factor D antibodyof humanized anti-Factor D #111, wherein the variable heavy chain domaincomprises the sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQID NO: 28 and SEQ ID NO: 29. In one example, the invention provides afragment of said anti-Factor D antibodies (e.g. antigen-bindingfragments).

In one aspect, the invention provides a modified anti-Factor D antibodyof humanized anti-Factor D #111, wherein the light chain domaincomprises the sequence of SEQ ID NO: 47. In another aspect, theinvention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the heavy chain domain comprises thesequence of SEQ ID NO: 54. In one aspect, the invention provides amodified anti-Factor D antibody of humanized anti-Factor D #111, whereinthe light chain domain comprises the sequence of SEQ ID NO: 61. Inanother aspect, the invention provides a modified anti-Factor D antibodyof humanized anti-Factor D #111, wherein the heavy chain domaincomprises the sequence of SEQ ID NO: 63. In one example, the inventionprovides a fragment of said anti-Factor D antibody (e.g. antigen-bindingfragment).

In one aspect, the invention provides a modified anti-Factor D antibodyof humanized anti-Factor D #111, wherein the variable light chain domaincomprises the sequence of SEQ ID NO: 6 and the variable heavy chaindomain comprises the sequence of SEQ ID NO: 18. In one embodiment, theinvention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the variable light chain domain comprisesthe sequence of SEQ ID NO: 7 and the variable heavy chain domaincomprises the sequence of SEQ ID NO: 19. In another embodiment, theinvention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the variable light chain domain comprisesthe sequence of SEQ ID NO: 8 and the variable heavy chain domaincomprises the sequence of SEQ ID NO: 20. In another embodiment, theinvention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the variable light chain domain comprisesthe sequence of SEQ ID NO: 9 and the variable heavy chain domaincomprises the sequence of SEQ ID NO: 21. In another embodiment, theinvention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the variable light chain domain comprisesthe sequence of SEQ ID NO: 10 and variable heavy chain domain comprisesthe sequence of SEQ ID NO: 22. In another embodiment, the inventionprovides a modified anti-Factor D antibody of humanized anti-Factor D#111, wherein the variable light chain domain comprises the sequence ofSEQ ID NO: 11 and the variable heavy chain domain comprises the sequenceof SEQ ID NO: 23. In another embodiment, the invention provides amodified anti-Factor D antibody of humanized anti-Factor D #111, whereinthe variable light chain domain comprises the sequence of SEQ ID NO: 12and the variable heavy chain domain comprises the sequence of SEQ ID NO:24. In another embodiment, the invention provides a modified anti-FactorD antibody of humanized anti-Factor D #111, wherein the variable lightchain domain comprises the sequence of SEQ ID NO: 13 and the variableheavy chain domain comprises the sequence of SEQ ID NO: 25. In anotherembodiment, the invention provides a modified anti-Factor D antibody ofhumanized anti-Factor D #111, wherein the variable light chain domaincomprises the sequence of SEQ ID NO: 14 and the variable heavy chaindomain comprises the sequence of SEQ ID NO: 26. In another embodiment,the invention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the variable light chain domain comprisesthe sequence of SEQ ID NO: 15 and the variable heavy chain domaincomprises the sequence of SEQ ID NO: 27. In another embodiment, theinvention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the variable light chain domain comprisesthe sequence of SEQ ID NO: 16 and the variable heavy chain domaincomprises the sequence of SEQ ID NO: 28. In another embodiment, theinvention provides a modified anti-Factor D antibody of humanizedanti-Factor D #111, wherein the variable light chain domain comprisesthe sequence of SEQ ID NO: 17 and the variable heavy chain domaincomprises the sequence of SEQ ID NO: 29. In one example, the inventionprovides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

In one aspect, the invention provides a modified anti-Factor D antibodyof humanized anti-Factor D #111, wherein the light chain domaincomprises the sequence of SEQ ID NO: 47 and the heavy chain domaincomprises the sequence of SEQ ID NO: 54. In one aspect, the inventionprovides a modified anti-Factor D antibody of humanized anti-Factor D#111, wherein the light chain domain comprises the sequence of SEQ IDNO: 61 and the heavy chain domain comprises the sequence of SEQ ID NO:63.

3. Affinity and Biological Activity of Anti-Factor D Antibodies

The invention herein includes antibodies, and variants thereof orfragments thereof (e.g. antigen-binding fragments), havingcharacteristics identified herein as being desireable in an anti-FactorD antibody. Antibodies, and variants thereof or fragments thereof (e.g.antigen-binding fragments), having characteristics identified herein asbeing desireable in an anti-Factor D antibody, may be screened forinhibitory biological activity, for example in vitro or in vivo, or bymeasuring binding affinity.

a. Affinity

To determine whether an anti-Factor D antibody, and variants thereof orfragments thereof (e.g. antigen-binding fragments), bind to the sameepitope on human Factor D bound by an antibody of interest (for example,those antibodies which antagonize Factor D activity), a cross-blockingassay may be performed (Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Ed Harlow and David Lane (1988)). Alternatively,epitope mapping may be performed to determine whether an anti-Factor Dantibody binds an epitope of interest (Champe et al., J. Biol. Chem.,270: 1388-1394 (1995). Antibody affinities, for example for human FactorD, may be determined using standard methods, including those describedin Example 3.

In one aspect, the invention provides anti-Factor D antibodies, orantibody variants thereof, or fragments thereof (e.g. antigen-bindingfragments), that compete with a murine anti-Factor D antibody and/orhumanized anti-Factor D antibody clone #111, and/or an antibodycomprising variable domain or HVR sequences of humanized anti-Factor Dantibody clone #111. Anti-Factor D antibodies, or variants thereof, orfragments thereof (e.g. antigen-binding fragments) that bind to the sameepitope as a murine anti-Factor D antibody and/or humanized anti-FactorD antibody clone #111, and/or an antibody comprising variable domain orHVR sequences of humanized anti-Factor D antibody clone #111, are alsoprovided.

In one embodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the monovalent affinity of theantibody to Factor D (e.g., affinity of the antibody as a Fab fragmentto Factor D) is lower, for example at least 1-fold or 2-fold lower thanthe monovalent affinity of a chimeric antibody (e.g. affinity of thechimeric antibody as a Fab fragment to Factor D), comprising, consistingor consisting essentially of a light chain variable domain of and heavychain variable domain from a murine anti-Factor D antibody. In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In one embodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the bivalent affinity of the antibodyto Factor D (e.g., affinity of the antibody as an IgG to Factor D) islower, for example at least 1-fold or 2-fold lower than the bivalentaffinity of a chimeric antibody (e.g. affinity of the chimeric antibodyas an IgG to Factor D), comprising, consisting or consisting essentiallyof a light chain variable domain and heavy chain variable domain from amurine anti-Factor D antibody. In one example, the invention provides afragment of said anti-Factor D antibodies (e.g. antigen-bindingfragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variant thereof, wherein the monovalent affinity of theantibody to Factor D (e.g., affinity of the antibody as a Fab fragmentto Factor D) is greater, for example at least 1-fold or 2-fold greaterthan the monovalent affinity of a chimeric antibody (e.g. affinity ofthe chimeric antibody as a Fab fragment to Factor D), comprising,consisting or consisting essentially of a light chain variable domainand heavy chain variable domain from a murine anti-Factor D antibody. Inone example, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variants thereof, wherein the bivalent affinity of theantibody to Factor D (e.g., affinity of the antibody as an IgG to FactorD) is greater, for example at least 1-fold or 2-fold greater than thebivalent affinity of a chimeric antibody (e.g. affinity of the chimericantibody as an IgG to Factor D), comprising, consisting or consistingessentially of a light chain variable domain and heavy chain variabledomain from a murine anti-Factor D antibody. In one example, theinvention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 20 nM (20×10⁻⁹ M) or better. In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 10 nM (10×10⁻⁹ M) or better. In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 1.0 nM (1.0×10⁻⁹ M) or better. In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 0.5 nM (0.5×10⁻⁹ M) or better. In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 1.0 μM (1.0×10⁻¹² M) or better. In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 0.5 μM (0.5×10⁻¹² M) or better. In one example,the invention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variant thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is 10.0 nM (10.0×10⁻⁹ M) or better. In another embodiment, theinvention provides an anti-Factor D antibody, or antibody variantthereof, wherein the affinity of the antibody in its bivalent form toFactor D (e.g., affinity of the antibody as an IgG to Factor D) is 5.0nM (5.0×10⁻⁹ M) or better. In another embodiment, the invention providesan anti-Factor D antibody, or antibody variant thereof, wherein theaffinity of the antibody in its bivalent form to Factor D (e.g.,affinity of the antibody as an IgG to Factor D) is 1.0 nM (1.0×10⁻⁹ M)or better. In another embodiment, the invention provides an anti-FactorD antibody, or antibody variant thereof, wherein the affinity of theantibody in its bivalent form to Factor D (e.g., affinity of theantibody as an IgG to Factor D) is 0.5 nM (0.5×10⁻⁹ M) or better. Inanother embodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is 5.0 μM (5.0×10⁻¹² M) or better. In another embodiment, theinvention provides an anti-Factor D antibody, or antibody variantthereof, wherein the affinity of the antibody in its bivalent form toFactor D (e.g., affinity of the antibody as an IgG to Factor D) is 2.0μM (2.0×10⁻¹² M) or better. In another embodiment, the inventionprovides an anti-Factor D antibody, or antibody variant thereof, whereinthe affinity of the antibody in its bivalent form to Factor D (e.g.,affinity of the antibody as an IgG to Factor D) is 1.0 μM (1.0×10⁻¹² M)or better. In another embodiment, the invention provides an anti-FactorD antibody, or antibody variant thereof, wherein the affinity of theantibody in its bivalent form to Factor D (e.g., affinity of theantibody as an IgG to Factor D) is 0.5 μM (0.5×10⁻¹² M) or better. Inone example, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is between 0.5 mM (0.5×10⁻⁶ M) and 0.5 μM(0.5×10⁻¹² M). In another embodiment, the invention provides ananti-Factor D antibody, or antibody variant thereof, wherein theaffinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is between 15 nM(15×10⁻⁹ M) and 0.1 nM (0.1×10⁻⁹ M). In another embodiment, theinvention provides an anti-Factor D antibody, or antibody variantthereof, wherein the affinity of the antibody in its monovalent form toFactor D (e.g., affinity of the antibody as a Fab fragment to Factor D)is between 5.5 nM (5.5×10⁻⁹ M) and 1 nM (1×10⁻⁹ M). In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is between 0.5 μM (0.5×10⁻¹² M) and 2 μM (2×10⁻¹²M). In one example, the invention provides a fragment of saidanti-Factor D antibodies (e.g. antigen-binding fragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variant thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is between 0.5 mM (0.5×10⁻⁶ M) and 0.5 pM (0.5×10⁻¹² M). Inanother embodiment, the invention provides an anti-Factor D antibody, orantibody variants thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is between 10 nM (10×10⁻⁹ M) and 0.05 nM (0.05×10⁻⁹ M). Inanother embodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is between 5.5 nM (5.5×10⁻⁹ M) and 1 nM (1×10⁻⁹ M). In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is between 0.5 μM (0.5×10⁻¹² M) and 2 μM (2×10⁻¹² M). In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is about 1.4 μM (1.4×10⁻¹² M). In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as a IgG toFactor D) is about 1.1 μM (1.1×10⁻¹² M). In another embodiment, theinvention provides an anti-Factor D antibody, or antibody variantthereof, wherein the affinity of the antibody in its monovalent form toFactor D (e.g., affinity of the antibody as a Fab fragment to Factor D)is about 0.19 nM (0.19×10⁻⁹ M). In another embodiment, the inventionprovides an anti-Factor D antibody, or antibody variant thereof, whereinthe affinity of the antibody in its bivalent form to Factor D (e.g.,affinity of the antibody as a IgG to Factor D) is about 0.08 nM(0.08×10⁻⁹ M). In another embodiment, the invention provides ananti-Factor D antibody, or antibody variant thereof, wherein theaffinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is about 12.3 nM(12.3×10⁻⁹ M). In another embodiment, the invention provides ananti-Factor D antibody, or antibody variant thereof, wherein theaffinity of the antibody in its bivalent form to Factor D (e.g.,affinity of the antibody as a IgG to Factor D) is about 9.0 nM (9.0×10⁻⁹M). In one example, the invention provides a fragment of saidanti-Factor D antibodies (e.g. antigen-binding fragments).

In another embodiment, the invention provides an anti-Factor D antibody,or antibody variant thereof, wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is about 1.4 μM (1.4×10⁻¹² M)±0.5. In anotherembodiment, the invention provides an anti-Factor D antibody, orantibody variant thereof, wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is about 1.1 μM (1.1×10⁻¹² M)±0.6. In another embodiment, theinvention provides an anti-Factor D antibody, or antibody variantthereof, wherein the affinity of the antibody in its monovalent form toFactor D (e.g., affinity of the antibody as a Fab fragment to Factor D)is about 0.19 nM (0.19×10⁻⁹ M)±0.01. In another embodiment, theinvention provides an anti-Factor D antibody, or antibody variantthereof, wherein the affinity of the antibody in its bivalent form toFactor D (e.g., affinity of the antibody as a IgG to Factor D) is about0.08 nM (0.08×10⁻⁹ M)±0.01. In another embodiment, the inventionprovides an anti-Factor D antibody, or antibody variant thereof, whereinthe affinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is about 12.3 nM(12.3×10⁻⁹ M)±2. In another embodiment, the invention provides ananti-Factor D antibody, or antibody variant thereof, wherein theaffinity of the antibody in its bivalent form to Factor D (e.g.,affinity of the antibody as a IgG to Factor D) is about 9.0 nM (9.0×10⁻⁹M)±1. In one example, the invention provides a fragment of saidanti-Factor D antibodies (e.g. antigen-binding fragments).

In another embodiment, an anti-Factor D antibody, or antibody variantthereof, may have an affinity in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) of about 1.4 pM(1.4×10⁻¹² M)±2. In another embodiment, an anti-Factor D antibody, orantibody variant thereof, may have an affinity in its bivalent form toFactor D (e.g., affinity of the antibody as a IgG to Factor D) of about1.1 μM (1.1×10⁻¹² M)±2. In another embodiment, an anti-Factor Dantibody, or antibody variant thereof, may have an affinity in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is about 0.19 nM (0.19×10⁻⁹ M)±2. In anotherembodiment, an anti-Factor D antibody, or antibody variant thereof, mayhave an affinity in its bivalent form to Factor D (e.g., affinity of theantibody as a IgG to Factor D) is about 0.08 nM (0.08×10⁻⁹ M)±2. Inanother embodiment, an anti-Factor D antibody, or antibody variantthereof, may have an affinity in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is about 12.3 nM(12.3×10⁻⁹ M)±2. In another embodiment, an anti-Factor D antibody, orantibody variant thereof, may have an affinity in its bivalent form toFactor D (e.g., affinity of the antibody as a IgG to Factor D) is about9.0 nM (9.0×10⁻⁹ M)±2. In one example, the invention provides a fragmentof said anti-Factor D antibodies (e.g. antigen-binding fragments).

As is well-established in the art, binding affinity of a ligand to itsreceptor can be determined using any of a variety of assays, andexpressed in terms of a variety of quantitative values. Accordingly, inone embodiment, the binding affinity is expressed as K_(D) values andreflects intrinsic binding affinity (e.g., with minimized avidityeffects). Generally and preferably, binding affinity is measured invitro, whether in a cell-free or cell-associated setting. As describedin greater detail herein, fold difference in binding affinity can bequantified in terms of the ratio of the monovalent binding affinityvalue of a humanized antibody (e.g., in Fab form) and the monovalentbinding affinity value of a reference/comparator antibody (e.g., in Fabform) (e.g., a murine antibody having donor hypervariable regionsequences), wherein the binding affinity values are determined undersimilar assay conditions. Thus, in one embodiment, the fold differencein binding affinity is determined as the ratio of the K_(D) values ofthe humanized antibody in Fab form and said reference/comparator Fabantibody. For example, in one embodiment, if an antibody of theinvention (A) has an affinity that is “3-fold lower” than the affinityof a reference antibody (M), then if the K_(D) value for A is 3×, theK_(D) value of M would be 1×, and the ratio of K_(D) of A to K_(D) of Mwould be 3:1. Conversely, in one embodiment, if an antibody of theinvention (C) has an affinity that is “3-fold greater” than the affinityof a reference antibody (R), then if the K_(D) value for C is 1×, theK_(D) value of R would be 3×, and the ratio of K_(D) of C to K_(D) of Rwould be 1:3. Any of a number of assays known in the art, includingthose described herein, can be used to obtain binding affinitymeasurements, including, for example, Biacore, radioimmunoassay (RIA)and ELISA.

Further, K_(D) values for an antibody of the invention may varydepending on conditions of the particular assay used. For example, inone embodiment, binding affinity measurements may be obtained in anassay wherein the Fab or antibody is immobilized and binding of theligand, i.e. Factor D, is measured or alternatively, the ligand, i.e.Factor D, for the Fab or antibody is immobilized and binding of the Fabor antibody is measured. In one embodiment, the binding affinitymeasurements may be obtained in an assay wherein the regenerationconditions may comprise (1) 10 mM glycine or 4M MgCl₂ at pH 1.5, and (2)pH between pH of 1.0 and pH of 7.5, including pH of 1.5, pH of 5.0, pHof 6.0 and pH of 7.2. In one embodiment, the binding affinitymeasurements may be obtained in an assay wherein the binding conditionsmay comprise (1) PBS or HEPES-buffered saline and (2) Tween-20, i.e.0.1% Tween-20. In one embodiment, the binding affinity measurements maybe obtained in an assay wherein the source of the ligand, i.e. Factor D,may be from commercially available sources. In one embodiment, bindingaffinity measurements may be obtained in an assay wherein (1) the Fab orantibody is immobilized and binding of the ligand, i.e. Factor D ismeasured, (2) the regeneration conditions comprise 4M MgCl₂ at pH 7.2and (3) the binding conditions comprise HEPES-buffered saline, pH 7.2containing 0.1% Tween-20. In one embodiment, binding affinitymeasurements may be obtained in an assay wherein (1) the ligand, i.e.Factor D, is immobilized and binding of the Fab or antibody is measured,(2) the regeneration conditions comprise 10 mM glycine at pH 1.5 and (3)the binding conditions comprise PBS buffer.

b. Biological Activity

To determine whether an anti-Factor D antibody, or variant or fragmentthereof (e.g. antigen-binding fragment) is capable of binding to FactorD and exerting a biological effect, for example, inhibition ofalternative pathway hemolysis, hemolytic inhibition assays using rabbitRBCs may be used, including those described in Example 2. Such hemolyticinhibition may be determined using standard assays (Kostavasili et al.,J of Immunology, 158(4): 1763-72 (1997); Wiesmann et al., Nature,444(7116): 159-60 (2006)). Activation of complement in such assays maybe initiated with serum or plasma. Appropriate concentrations of FactorD in serum or plasma (Pascual et al., Kidney International, 34: 529-536(1998); Complement Facts Book, Bernard J. Morley and Mark J. Walport,editors, Academic Press (2000); Barnum et al., J. Immunol. Methods, 67:303-309 (1984)) can be routinely determined according to methods knownin the art, including those that have been described in references suchas Pascual et al., Kidney International, 34: 529-536 (1998) and Barnumet al., J. Immunol. Methods, 67: 303-309 (1984) and Example 4. Thepresent invention relates generally to antibodies capable of inhibitingbiological activities associated with Factor D. For example, at aconcentration of 18 μg/ml (equivalent to about 1.5 times the molarconcentration of human factor D in the blood; molar ratio of anti-FactorD antibody to Factor D of about 1.5:1), significant inhibition of thealternative complement activity by the antibody can be observed (see,e.g., U.S. Pat. No. 6,956,107)

In one embodiment, the invention includes anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with IC₅₀ values less than 30 nM. In one embodiment, theinvention includes anti-Factor D antibodies, wherein a Fab fragment ofsuch antibodies inhibits alternative pathway hemolysis with IC₅₀ valuesless than 15 nM. In one embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibits, inhibitsalternative pathway hemolysis with IC₅₀ values less than 10 nM. In oneembodiment, the invention provides anti-Factor D antibodies, wherein aFab fragment of such antibodies inhibits alternative pathway hemolysiswith IC₅₀ values less than 5 nM. In one example, the invention providesa fragment of said anti-Factor D antibodies (e.g. antigen-bindingfragments).

In one embodiment, the invention provides anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with IC₅₀ values between 30 nM and 2 nM. In one embodiment,the invention provides anti-Factor D antibodies, wherein a Fab fragmentof such antibodies inhibits alternative pathway hemolysis with IC₅₀values between 25 nM and 7 nM. In one embodiment, the invention providesanti-Factor D antibodies, wherein a Fab fragment of such antibodiesinhibits alternative pathway hemolysis with IC₅₀ values between 20 nMand 12 nM. In one embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway hemolysis with IC₅₀ values between 30 nM and 15 nM.In one embodiment, the invention provides anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with IC₅₀ values between 12 nM and 8 nM. In one embodiment,the invention provides anti-Factor D antibodies, wherein a Fab fragmentof such antibodies inhibits alternative pathway hemolysis with IC₅₀values between 7 nM and 2 nM. In one embodiment, the invention providesanti-Factor D antibodies, wherein a Fab fragment of such antibodiesinhibits alternative pathway hemolysis with IC₅₀ values between 6 nM and3 nM. In one embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway hemolysis with IC₅₀ values between 8 nM and 5 nM. Inone embodiment, the invention provides anti-Factor D antibodies, whereina Fab fragment of such antibodies inhibits alternative pathway hemolysiswith IC₅₀ values between 5 nM and 2 nM. In one embodiment, the inventionprovides anti-Factor D antibodies, wherein a Fab fragment of suchantibodies inhibits alternative pathway hemolysis with IC₅₀ valuesbetween 10 nM and 5 nM. In one embodiment, the invention providesanti-Factor D antibodies, wherein a Fab fragment of such antibodiesinhibits alternative pathway hemolysis with IC₅₀ values between 8 nM and2 nM. In one embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway hemolysis with IC₅₀ values between 7 nM and 3 nM. Inone embodiment, the invention provides anti-Factor D antibodies, whereina Fab fragment of such antibodies inhibits alternative pathway hemolysiswith IC₅₀ values between 6 nM and 4 nM. In another embodiment, theinvention provides anti-Factor D antibodies, wherein a Fab fragment ofsuch antibodies inhibits alternative pathway hemolysis with an IC₅₀value of about 4.7 nM±0.6 nM. In another embodiment, the inventionprovides anti-Factor D antibodies, wherein a Fab fragment of suchantibodies inhibits alternative pathway hemolysis with an IC₅₀ value ofabout 6.4 nM±0.6 nM. In another embodiment, the invention providesanti-Factor D antibodies, wherein a Fab fragment of such antibodiesinhibits alternative pathway hemolysis with an IC₅₀ value of about 3.5nM±0.5 nM. In another embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway hemolysis with an IC₅₀ value of about 4.4 nM±1.5 nM.In another embodiment, the invention provides anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with an IC₅₀ value of about 10.2 nM±0.8 nM. In anotherembodiment, the invention provides anti-Factor D antibodies, wherein aFab fragment of such antibodies inhibits alternative pathway hemolysiswith an IC₅₀ value of about 23.9 nM±5.0 nM. In one example, theinvention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

In one embodiment, the invention provides anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with IC₉₀ values less than 80 nM. In one embodiment, theinvention provides anti-Factor D antibodies, wherein a Fab fragment ofsuch antibodies inhibits alternative pathway hemolysis with IC₉₀ valuesless than 50 nM. In one embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway hemolysis with IC₉₀ values less than 40 nM. In oneembodiment, the invention provides anti-Factor D antibodies, wherein aFab fragment of such antibodies inhibits alternative pathway hemolysiswith IC₉₀ values less than 20 nM. In one embodiment, the inventionprovides anti-Factor D antibodies, wherein a Fab fragment of suchantibodies inhibits alternative pathway hemolysis with IC₅₀ values lessthan 15 nM. In one example, the invention provides a fragment of saidanti-Factor D antibodies (e.g. antigen-binding fragments).

In one embodiment, the invention provides anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with IC₉₀ values between 80 nM and 10 nM. In one embodiment,the invention provides anti-Factor D antibodies, wherein a Fab fragmentof such antibodies inhibits alternative pathway hemolysis with IC₉₀values between 75 nM and 15 nM. In one embodiment, the inventionprovides anti-Factor D antibodies, wherein a Fab fragment of suchantibodies inhibits alternative pathway hemolysis with IC₉₀ valuesbetween 70 nM and 20 nM. In one embodiment, the invention providesanti-Factor D antibodies, wherein a Fab fragment of such antibodiesinhibits alternative pathway hemolysis with IC₉₀ values between 65 nMand 25 nM. In one embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway hemolysis with IC₉₀ values between 60 nM and 30 nM.In one embodiment, the invention provides anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with IC₉₀ values between 55 nM and 35 nM. In one embodiment,the invention provides anti-Factor D antibodies, wherein a Fab fragmentof such antibodies inhibits alternative pathway hemolysis with IC₉₀values between 50 nM and 40 nM. In one embodiment, the inventionprovides anti-Factor D antibodies, wherein a Fab fragment of suchantibodies inhibits alternative pathway hemolysis with IC₉₀ valuesbetween 80 nM and 70 nM. In one embodiment, the invention providesanti-Factor D antibodies, wherein a Fab fragment of such antibodiesinhibits alternative pathway hemolysis with IC₉₀ values between 55 nMand 25 nM. In one embodiment, the invention provides anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway hemolysis with IC₉₀ values between 16 nM and 12 nM.In another embodiment, the invention provides anti-Factor D antibodies,wherein a Fab fragment of such antibodies inhibits alternative pathwayhemolysis with an IC₉₀ value of about 14.0 nM±1.0 nM. In anotherembodiment, the invention provides anti-Factor D antibodies, wherein aFab fragment of such antibodies inhibits alternative pathway hemolysiswith an IC₉₀ value of about 38.0 nM±11.0 nM. In another embodiment, theinvention provides anti-Factor D antibodies, wherein a Fab fragment ofsuch antibodies inhibits alternative pathway hemolysis with an IC₉₀value of about 72.6 nM±4.8 nM. In one example, the invention provides afragment of said anti-Factor D antibodies (e.g. antigen-bindingfragments).

In one embodiment, the invention concerns an anti-Factor D antibody, orfragment thereof (e.g. antigen-binding fragment) wherein a Fab fragmentof such antibodies inhibits alternative pathway hemolysis in an antibodyto Factor D molar ratio of about 0.05:1 (0.05) to about 10:1 (10), orabout 0.09:1 (0.09) to about 8:1 (8), or about 0.1:1 (0.1) to about 6:1(6), or about 0.15:1 (0.15) to about 5:1 (5), or about 0.19:1 (0.19) toabout 4:1 (4), or about 0.2:1 (0.2) to about 3:1 (3), or about 0.3:1(0.3) to about 2:1 (2), or about 0.4:1 (0.4) to about 1:1 (1), or about0.5:1 (0.5) to about 1:2 (0.5), or about 0.6:1 (0.6) to about 1:3(0.33), or about 0.7:1 (0.7) to about 1:4 (0.25), or about 0.8:1 (0.8)to about 1:5 (0.2) or about 0.9:1 (0.9) to about 1:6 (0.17). In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In one embodiment, the present invention includes fragments of humanizedanti-Factor D antibodies (e.g. antigen-binding fragments). The antibodyfragments of the present invention may, for example, be Fab, Fab′,F(ab′)₂, scFv, (scFv)₂, dAb, complementarity determining region (CDR)fragments, linear antibodies, single-chain antibody molecules,minibodies, diabodies, or multispecific antibodies formed from antibodyfragments. In a further embodiment, the invention provides a humanizedanti-Factor D antibody fragment (e.g. antigen-binding fragment) that iscapable of penetrating substantially all of the retina. In an evenfurther embodiment, the invention provides a humanized anti-Factor Dantibody fragment (e.g. antigen-binding fragment) that is capable ofpenetrating throughout the entire thickness of the retina. In oneexample, the invention provides a fragment of said anti-Factor Dantibodies (e.g. antigen-binding fragments).

In one embodiment, the present invention includes humanized anti-FactorD antibodies, wherein a Fab fragment of such antibodies have a half lifeof at least 3, 5, 7, 10 or 12 days after administration into a mammalianeye (e.g. human) via a single intravitreal injection. In anotherembodiment, the present invention includes humanized anti-Factor Dantibodies, wherein a Fab fragment of such antibodies inhibitsalternative pathway (AP) complement activation for at least 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110 or 115 days afteradministration into a mammalian eye (e.g. human) via a singleintravitreal injection. In another embodiment, the present inventionincludes humanized anti-Factor D antibodies, wherein the concentrationof a Fab fragment of such antibodies that inhibits alternative pathway(AP) complement activation is maintained in retinal tissue for at least40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 days after administration intoa mammalian eye (e.g. human) via a single intravitreal injection. Inanother embodiment, the present invention includes humanized anti-FactorD antibodies, wherein the concentration of a Fab fragment of suchantibodies that inhibits alternative pathway (AP) complement activationis maintained in the vitreous humor for at least 80, 85, 90, 95, 100,105, 110 or 115 days after administration into a mammalian eye (e.g.human) via a single intravitreal injection. In one example, theinvention provides a fragment of said anti-Factor D antibodies (e.g.antigen-binding fragments).

Generation of Antibodies

Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for anti-Factor D antibody production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: a Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation, which means introduction of DNA into the host so thatthe DNA is replicable, either as an extrachromosomal or by chromosomalintegrant, are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated, polyethylene-gycol/DMSO andelectroporation. Depending on the host cell used, transformation isperformed using standard techniques appropriate to such cells. Thecalcium treatment employing calcium chloride, as described in Sambrooket al., supra, or electroporation is generally used for prokaryotes.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published 29 Jun. 1989. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham and vander Eb, Virology, 52:456-457 (1978) can be employed. General aspects ofmammalian cell host system transfections have been described in U.S.Pat. No. 4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are for cloning or expressing the DNA in the vectors herein areprokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes forthis purpose include both Gram-negative and Gram-positive organisms, forexample, Enterobacteria such as Escherichia, e.g. E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli,such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas, such as P.aeruginosa, Rhizobia, Vitreoscilla, Paracoccus, and Streptomyces. Onepreferred E. coli cloning host is E. coli 294 (ATCC 31,446), althoughother strains such as E. coli B, E. coli X1776 (ATCC 31,537), E. coliW3110 (ATCC 27,325) and K5 772 (ATCC 53,635) are suitable. Theseexamples are illustrative rather than limiting. Strain W3110 is oneparticularly preferred host or parent host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell secretes minimal amounts of proteolytic enzymes. For example,strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2(Washington, D.C.: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No. 27,325) may be modified to effect a geneticmutation in the genes encoding proteins endogenous to the host, withexamples of such hosts including E. coli W3110 strain 1 A2, which hasthe complete genotype tonA; E. coli W3110 strain 9E4, which has thecomplete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244),which has the complete genotype tonA ptr3phoA E15 (argF-lac)169 degPompT kan′; E. coli W3110 strain 37D6, which has the complete genotypetonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan^(r) ; E. coliW3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistantdegP deletion mutation; E. coli W3110 strain 33D3 having genotype W3110ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kan^(R) (U.S.Pat. No. 5,639,635) and an E. coli strain having mutant periplasmicprotease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Otherstrains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E.coli B, E. coli _(λ) 1776 (ATCC 31,537) and E. coli RV308(ATCC 31,608)are also suitable. These examples are illustrative rather than limiting.Methods for constructing derivatives of any of the above-mentionedbacteria having defined genotypes are known in the art and described in,for example, Bass et al., Proteins, 8:309-314 (1990). It is generallynecessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. Coli, Serratia, or Salmonella species can be suitably used as thehost when well known plasmids such as pBR322, pBR325, pACYC177, orpKN410 are used to supply the replicon. Typically the host cell shouldsecrete minimal amounts of proteolytic enzymes, and additional proteaseinhibitors may desirably be incorporated in the cell culture.Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

Full length antibody, antibody fragments (e.g. antigen-bindingfragments), and antibody fusion proteins can be produced in bacteria, inparticular when glycosylation and Fc effector function are not needed,such as when the therapeutic antibody is conjugated to a cytotoxic agent(e.g., a toxin) and the immunoconjugate by itself shows effectiveness intumor cell destruction. Full length antibodies have greater half life incirculation. Production in E. coli is faster and more cost efficient.For expression of antibody fragments and polypeptides in bacteria, see,e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199(Joly et al.), and U.S. Pat. No. 5,840,523 (Simmons et al.) whichdescribes translation initiation region (TIR) and signal sequences foroptimizing expression and secretion, these patents incorporated hereinby reference. After expression, the antibody is isolated from the E.coli cell paste in a soluble fraction and can be purified through, e.g.,a protein A or G column depending on the isotype. Final purification canbe carried out similar to the process for purifying antibody expressede.g, in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as Schizosaccharomyces pombe; Kluyveromyces; Candida;Trichoderma; Neurospora crassa; and filamentous fungi such as e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such asA. nidulans and A. niger. Methylotropic yeasts are suitable herein andinclude, but are not limited to, yeast capable of growth on methanolselected from the genera consisting of Hansenula, Candida, Kloeckera,Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specificspecies that are exemplary of this class of yeasts may be found in C.Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells, Luckow et al., Bio/Technology 6, 47-55 (1988); Miller etal., Genetic Engineering, Setlow et al. eds. Vol. 8, pp. 277-279 (Plenampublishing 1986); Mseda et al., Nature 315, 592-594 (1985). Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Moreover, plant cellscultures of cotton, corn, potato, soybean, petunia, tomato, and tobaccoand also be utilized as hosts.

Vertebrate cells, and propagation of vertebrate cells, in culture(tissue culture) has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, eds. (1973). Examples of usefulmammalian host cell lines are monkey kidney; human embryonic kidneyline; baby hamster kidney cells; Chinese hamster ovary cells/-DHFR (CHO,Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mousesertoli cells; human cervical carcinoma cells (HELA); canine kidneycells; human lung cells; human liver cells; mouse mammary tumor; and NS0cells.

For recombinant production of an antibody of the invention, orantibody-fragment thereof (e.g. antigen-binding fragment), the nucleicacid (e.g., cDNA or genomic DNA) encoding it is isolated and insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. DNA encoding the antibody, or fragment thereof (e.g.antigen-binding fragment) is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin.

The vector may, for example, be in the form of a plasmid, cosmid, viralparticle, or phage. The appropriate nucleic acid sequence may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan.

The Factor D may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe anti-Factor D antibody-encoding DNA that is inserted into thevector. The signal sequence may be a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeastsecretion the signal sequence may be, e.g., the yeast invertase leader,alpha factor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 362,179published 4 Apr. 1990), or the signal described in WO 90/13646 published15 Nov. 1990. In mammalian cell expression, mammalian signal sequencesmay be used to direct secretion of the protein, such as signal sequencesfrom secreted polypeptides of the same or related species, as well asviral secretory leaders.

Host cells are transformed with the above-described vectors for antibodyproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

The host cells used to produce the antibody, or antibody variant orfragment (e.g. antigen-binding fragment) thereof, of this invention maybe cultured in a variety of media.

a. Prokaryotic Host Cells

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

b. Eukaryotic Host Cells

Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium (DMEM, Sigma) are suitable for culturing hostcells. In addition, any of the media described in Ham et al., Meth.Enzymol. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980),U.S. Pat. Nos. 4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; or6,048,728 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as X-chlorides, where X is sodium, calcium, magnesium; andphosphates), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Antibody Purification

Forms of anti-Factor D antibodies, or fragments thereof (e.g.antigen-binding fragments) may be recovered from culture medium or fromhost cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or byenzymatic cleavage. Cells employed in expression of anti-Factor Dantibody can be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify anti-Factor D antibody from recombinant cellproteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the anti-Factor D antibody. Various methods ofprotein purification may be employed and such methods are known in theart and described for example in Deutscher, Methods in Enzymology, 182(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected willdepend, for example, on the nature of the production process used andthe particular anti-Factor D antibody produced.

When using recombinant techniques, the antibody, or antibody variant orfragment (e.g. antigen-binding fragment) thereof, can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody, or antibody variant or fragment (e.g.antigen-binding fragment) thereof, is produced intracellularly, as afirst step, the particulate debris, either host cells or lysedfragments, may be removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody, or antibodyvariant or fragment (e.g. antigen-binding fragment) thereof, is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel elecrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing one purification technique. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody, or antibody variant orfragment (e.g. antigen-binding fragment) thereof. Protein A can be usedto purify antibodies that are based on human IgG1, IgG2 or IgG4 heavychains (Lindmark et al., J. Immunol Meth. 62: 1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human IgG3 (Guss et al., EMBOJ. 5: 1567-1575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibody,or antibody variant or fragment (e.g. antigen-binding fragment) thereof,comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody, or antibody variant or fragment(e.g. antigen-binding fragment) thereof, to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody, or antibody variant or fragment (e.g. antigen-bindingfragment) thereof, of interest and contaminants may be subjected to lowpH hydrophobic interaction chromatography using an elution buffer at apH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Therapeutic formulations of the polypeptide or antibody, or antibodyfragment thereof (e.g. antigen-binding fragment), or antibody variantthereof, may be prepared for storage as lyophilized formulations oraqueous solutions by mixing the polypeptide having the desired degree ofpurity with optional “pharmaceutically-acceptable” carriers, excipientsor stabilizers typically employed in the art (all of which are termed“excipients”). For example, buffering agents, stabilizing agents,preservatives, isotonifiers, non-ionic detergents, antioxidants andother miscellaneous additives. (See Remington's Pharmaceutical Sciences,16th edition, A. Osol, Ed. (1980)). Such additives must be nontoxic tothe recipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, there may be mentioned phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, and 3-pentanol.

Isotonicifiers sometimes known as “stabilizers” may be added to ensureisotonicity of liquid compositions of the present invention and includepolhydric sugar alcohols, preferably trihydric or higher sugar alcohols,such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,.alpha.-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (i.e. <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; polysaccharides such as dextran.Stabilizers may be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation herein mayalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desireable to further provide an immunosuppressive agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The active ingredients may also beentrapped in microcapsule prepared, for example, by coascervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin micropheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, A. Osal, Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished, for example, by filtration through sterilefiltration membranes. Sustained-release preparations may be prepared.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theantibody, or antibody variant or fragment (e.g. antigen-bindingfragment) thereof, which matrices are in the form of shaped articles,e.g., films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C. resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The compounds of the invention for prevention or treatment of an oculardisease or condition are typically administered by ocular, intraocular,and/or intravitreal injection, and/or juxtascleral injection, and/orsubtenon injection, and/or superchoroidal injection and/or topicaladministration in the form of eyedrops and/or ointment. Such compoundsof the invention may be delivered by a variety of methods, e.g.intravitreally as a device and/or a depot that allows for slow releaseof the compound into the vitreous, including those described inreferences such as Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, andPearson, editors, Taylor & Francis (March 2006). In one example, adevice may be in the form of a minpump and/or a matrix and/or a passivediffusion system and/or encapsulated cells that release the compound fora prolonged period of time (Intraocular Drug Delivery, Jaffe, Jaffe,Ashton, and Pearson, editors, Taylor & Francis (March 2006). Othermethods of administration may also be used, which includes but is notlimited to, topical, parenteral, subcutaneous, intraperitoneal,intrapulmonary, intranasal, and intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration.

Formulations for ocular, intraocular or intravitreal administration canbe prepared by methods and using ingredients known in the art. A mainrequirement for efficient treatment is proper penetration through theeye. Unlike diseases of the front of the eye, where drugs can bedelivered topically, retinal diseases require a more site-specificapproach. Eye drops and ointments rarely penetrate the back of the eye,and the blood-ocular barrier hinders penetration of systemicallyadministered drugs into ocular tissue. Accordingly, usually the methodof choice for drug delivery to treat retinal disease, such as AMD andCNV, is direct intravitreal injection. Intravitrial injections areusually repeated at intervals which depend on the patient's condition,and the properties and half-life of the drug delivered. For intraocular(e.g. intravitreal) penetration, usually molecules of smaller size arepreferred.

The efficacy of the treatment of complement-associated eye conditions,such as AMD or CNV, can be measured by various endpoints commonly usedin evaluating intraocular diseases. For example, vision loss can beassessed. Vision loss can be evaluated by, but not limited to, e.g.,measuring by the mean change in best correction visual acuity (BCVA)from baseline to a desired time point (e.g., where the BCVA is based onEarly Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chartand assessment at a test distance of 4 meters), measuring the proportionof subjects who lose fewer than 15 letters in visual acuity at a desiredtime point compared to baseline, measuring the proportion of subjectswho gain greater than or equal to 15 letters in visual acuity at adesired time point compared to baseline, measuring the proportion ofsubjects with a visual-acuity Snellen equivalent of 20/2000 or worse ata desired time point, measuring the NEI Visual FunctioningQuestionnaire, measuring the size of CNV and amount of leakage of CNV ata desired time point, e.g., by fluorescein angiography, etc. Ocularassessments can be done, e.g., which include, but are not limited to,e.g., performing eye exam, measuring intraocular pressure, assessingvisual acuity, measuring slitlamp pressure, assessing intraocularinflammation, etc.

The amount of therapeutic polypeptide, antibody, or antibody variantthereof, or fragment thereof (e.g antigen-binding fragment) which willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. Where possible, it is desirable todetermine the dose-response curve and the pharmaceutical compositions ofthe invention first in vitro, and then in useful animal model systemsprior to testing in humans.

In one embodiment, an aqueous solution of therapeutic polypeptide,antibody, or antibody variant thereof, or fragment thereof (e.g.antigen-binding fragment), is administered by subcutaneous injection. Inanother embodiment, an aqueous solution of therapeutic polypeptide,antibody, or antibody variant thereof, or fragment thereof (e.g.antigen-binding fragment) is administered by intravitreal injection.Each dose may range from about 0.5 μg to about 50 μg per kilogram ofbody weight, or more preferably, from about 3 μg to about 30 μg perkilogram body weight.

The dosing schedule for subcutaneous administration may vary form once amonth to daily depending on a number of clinical factors, including thetype of disease, severity of disease, and the subject's sensitivity tothe therapeutic agent.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby expressly incorporated by reference in their entirety.

Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacturecontaining materials useful for the treatment, prevention and/ordiagnosis of conditions targeted by the antibodies of the invention, orvariants thereof or fragments thereof (e.g. antigen-binding fragments).For example, the invention concerns an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis ofcomplement-associated disorders. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for treating, preventing and/or diagnosis of thecomplement-associated condition and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-Factor D antibody, orfragment thereof (e.g. antigen-binding fragment) of the invention. Thelabel or package insert indicates that the composition is useful fortreatment, prevention and/or diagnosis of a particular condition.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In oneembodiment, the label or package insert indicates that the compositionis used for treating complement-associated disorders, such as, forexample, any of the conditions listed before, including eye disorderse.g. iage-related macular degeneration (AMD). The label or packageinsert will further comprise instructions for administering the antibodycomposition to the patient.

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

In another embodiment, kits are also provided that are useful forvarious purposes, e.g., for treatment, prevention and/or diagnosis ofcomplement-associated disorders, for complement-associated hemolysisassays, for purification or immunoprecipitation of Factor D polypeptidefrom cells. For isolation and purification of Factor D polypeptide, thekit can contain an anti-Factor D antibody coupled to beads (e.g.,sepharose beads). Kits can be provided which contain the antibodies fordetection and quantitation of Factor D polypeptide in vitro, e.g., in anELISA or a Western blot. As with the article of manufacture, the kitcomprises a container and a label or package insert on or associatedwith the container. The container holds a composition comprising atleast one anti-Factor antibody, or fragment thereof (e.g.antigen-binding fragment) of the invention. Additional containers may beincluded that contain, e.g., diluents and buffers, control antibodies.The label or package insert may provide a description of the compositionas well as instructions for the intended in vitro or detection use. Thelabel or package insert may provide instructions for the administration(e.g. the antibody, or antibody fragment thereof (e.g. antigen-bindingfragment) to a subject.

Uses for the Humanized Antibody

The humanized antibodies, or fragments thereof (e.g. antigen-bindingfragments) or variant thereof, of the present invention are useful indiagnostic assays, e.g., for detecting expression of a target ofinterest in specific cells, tissues, or serum. For diagnosticapplications, the antibody, or antibody variant thereof or fragmentthereof (e.g. antigen-binding fragment), typically will be labeled witha detectable moiety. Numerous labels are available. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for Use inEnzyme Immunoassay, in Methods in Enzym. (Ed. J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

Sometimes, the label is indirectly conjugated with the antibody, orantibody variant thereof or fragment thereof (e.g. antigen-bindingfragment). The skilled artisan will be aware of various techniques forachieving this. For example, the antibody, or antibody variant thereofor fragment thereof (e.g. antigen-binding fragment), can be conjugatedwith biotin and any of the three broad categories of labels mentionedabove can be conjugated with avidin, or vice versa. Biotin bindsselectively to avidin and thus, the label can be conjugated with theantibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), in this indirect manner. Alternatively, toachieve indirect conjugation of the label with the antibody, or antibodyvariant thereof or fragment thereof (e.g. antigen-binding fragment), theantibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), is conjugated with a small hapten (e.g.digloxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten antibody, or antibody variant thereof(e.g. anti-digloxin antibody) or fragment thereof (e.g. antigen-bindingfragment). Thus, indirect conjugation of the label with the antibody, orantibody variant thereof or fragment thereof (e.g. antigen-bindingfragment), can be achieved.

In another embodiment of the invention, the antibody, or antibodyvariant thereof or fragment thereof (e.g. antigen-binding fragment),need not be labeled, and the presence thereof can be detected using alabeled antibody which binds to the antibody, or antibody variantthereof or fragment thereof (e.g. antigen-binding fragment).

The antibodies, or antibody variants thereof, or fragment thereof (e.g.antigen-binding fragment) of the present invention may be employed inany known assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment). The amount of target in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remain unbound.

Sandwich assays involve the use of two antibodies, or fragments thereof(e.g. antigen-binding fragments) each capable of binding to a differentimmunogenic portion, or epitope, or the protein to be detected. In asandwich assay, the test sample to be analyzed is bound by a firstantibody which is immobilized on a solid support, and thereafter asecond antibody binds to the test sample, thus forming an insolublethree-part complex. See e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies, or antibody variants thereof, or fragments thereof (e.g.antigen-binding fragments) may also be used for in vivo diagnosticassays. Generally, the antibody, or antibody variant thereof or fragmentthereof (e.g. antigen-binding fragment), is labeled with aradionucleotide (such as .sup.111 In, .sup.99 Tc, .sup.14 C, .sup.131 I,.sup.3H, .sup.32 P or .sup.35 S) so that the tumor can be localizedusing immunoscintiography. For example, a high affinity anti-IgEantibody of the present invention may be used to detect the amount ofIgE present in, e.g., the lungs of an asthmatic patient.

The antibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), of the present invention can be provided in akit, i.e., packaged combination of reagents in predetermined amountswith instructions for performing the diagnostic assay. Where theantibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), is labeled with an enzyme, the kit mayinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

In Vivo uses for the Antibody

It is contemplated that the antibodies, or antibodies thereof, orfragments thereof (e.g. antigen-binding fragments) of the presentinvention may be used to treat a mammal. In one embodiment, theantibody, or antibody thereof, is administered to a nonhuman mammal forthe purposes of obtaining preclinical data, for example. Exemplarynonhuman mammals to be treated include nonhuman primates, dogs, cats,rodents and other mammals in which preclinical studies are performed.Such mammals may be established animal models for a disease to betreated with the antibody, or antibody thereof, or may be used to studytoxicity of the antibody of interest. In each of these embodiments, doseescalation studies may be performed on the mammal.

The antibody, or variant thereof, or fragment thereof (e.g.antigen-binding fragment) or polypeptide is administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theantibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), is suitably administered by pulse infusion,particularly with declining doses of the antibody, or antibody variantthereof or fragment thereof (e.g. antigen-binding fragment). Preferablythe dosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), or polypeptide will depend on the type ofdisease to be treated, the severity and course of the disease, whetherthe antibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment) and the discretion of the attending physician.

Depending on the type and severity of the disease, about 0.1 mg/kg to150 mg/kg (e.g., 0.1-20 mg/kg) of antibody, or antibody variant thereofor fragment thereof (e.g. antigen-binding fragment), is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 mg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays. An exemplary dosing regimen is disclosed in WO 94/04188.

The antibody compositions may be formulated, dosed and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody, or antibody variantthereof or fragment thereof (e.g. antigen-binding fragment), to beadministered will be governed by such considerations, and is the minimumamount necessary to prevent, ameliorate, or treat a disease or disorder.The antibody, or antibody variant thereof or fragment thereof (e.g.antigen-binding fragment), need not be, but is optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question. The effective amount of such other agents depends on theamount of antibody, or antibody variant thereof or fragment thereof(e.g. antigen-binding fragment), present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

The antibodies, or antibody variants thereof, or fragments thereof (e.g.antigen-binding fragments) of the present invention which recognizeFactor D as their target may be used to treat complement-mediateddisorders. These disorders are associated with excessive or uncontrolledcomplement activation. They include: Complement activation duringcardiopulmonary bypass operations; complement activation due toischemia-reperfusion following acute myocardial infarction, aneurysm,stroke, hemorrhagic shock, crush injury, multiple organ failure,hypobolemic shock and intestinal ischemia. These disorders can alsoinclude disease or condition is an inflammatory condition such as severeburns, endotoxemia, septic shock, adult respiratory distress syndrome,hemodialysis; anaphylactic shock, severe asthma, angioedema, Crohn'sdisease, sickle cell anemia, poststreptococcal glomerulonephritis andpancreatitis. The disorder may be the result of an adverse drugreaction, drug allergy, IL-2 induced vascular leakage syndrome orradiographic contrast media allergy. It also includes autoimmune diseasesuch as systemic lupus erythematosus, myasthenia gravis, rheumatoidarthritis, Alzheimer's disease and multiple sclerosis. Complementactivation is also associated with transplant rejection. Recently therehas been a strong correlation shown between complement activation andocular diseases such as age-related macular degeneration, diabeticretinopathy and other ischemia-related retinopathies, choroidalneovascularization (CNV), uveitis, diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, and retinalneovascularization.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation. Commercially available reagents referred to in theexamples were used according to manufacturer's instructions unlessotherwise indicated. The source of those cells identified in thefollowing examples, and throughout the specification, by ATCC accessionnumbers is the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209.

Example 1 Modification of Anti-Factor D Abs

To identify modified anti-Factor D antibodies, and variants thereof, andfragments thereof (e.g. antigen-binding fragments) that would havecommercially desireable characteristics such as homogeneity duringmanufacturing and production or for purposes of analyticalcharacterization, a site-directed mutagenesis approach was used togenerate modified humanized anti-factor D antibodies, and variantsthereof, and fragments thereof (e.g. antigen-binding fragments). First,the variable heavy and light chain domains from humanized anti-Factor DFab clone #111 (SEQ ID NO: 2 and SEQ ID NO: 1, respectively) weresubcloned into an expression plasmid. Secondly, oligonucleotidesencoding single mutations were annealed to the resulting expressionplasmid to introduce the site-directed mutations.

Initially, the variable heavy and light chain domains of humanizedanti-Factor D Fab clone #111 were subcloned into the plasmid pAEP1(pAEP1 is a plasmid for the expression of Fab antibodies in E. coli.),with the subcloning resulting in the introduction of a valine (V) atposition 104 (according to Kabat numbering, see FIG. 10) of the variablelight chain domain.

The subcloning of the variable light chain domain domain of humanizedanti-Factor D Fab clone #111 into pAEP1 involved the ligation of two DNAfragments. The first fragment was the pAEP1 vector in which the smallEcoRV/KpnI fragment had been removed. The second fragment was anapproximately 300 base pair EcoRV-KpnI PCR fragment generated from thelight chain plasmid for the humanized anti-Factor D Fab clone #111,using the following primers:

(SEQ ID NO: 67) 5′-TTTCCCTTTGATATCCAGGTGACCCAGTCTCCATCCT-3′ (SEQ ID NO:68) 5′-TTTCCCTTTGGTACCCTGGCCAAACGTGTACGGCAAAGAATC-3′.The subcloning of the variable light chain domain of humanizedanti-Factor D clone #111 into pAEP1 introduced a valine (V) at position104 because position 104 is 2 amino acids downstream of the restrictionendonuclease sites, EcoRV and KpnI, which were used to insert thevariable light chain domain of humanized anti-Factor D clone #111 intopAEP1 and therefore in the backbone of the pAEP1 plasmid. This resultingintermediate plasmid is herein referred to as “pAEP1-283-VL”.

The subcloning of the variable heavy chain domain of humanizedanti-Factor D clone #111 into pAEP1-238-VL involved the ligation of twoDNA fragments. The first fragment was the pAEP1-238-VL vector in whichthe small BsiWI/PspOMI fragment had been removed. The second fragmentwas an approximately 364 base pair BsiWI-PspOMI PCR fragment generatedfrom the heavy chain plasmid for the humanized anti-Factor D Fab clone#111, using the following primers:

(SEQ ID NO: 69) 5′-TTTGGGTTTCGTACGCTCAGGTCCAGCTGGTGCAATCTGGG-3′ (SEQ IDNO: 70) 5′-TTTGGGTTTGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCAGGG T-3′.This ligation of the two DNA fragments resulted in the plasmid forhumanized anti-Factor D Fab antibody variant 238 (also herein referredto as “238”; plasmid is herein referred to as “p238”).

After the subcloning of the variable light and heavy chain domains fromhumanized anti-Factor D #111, site-directed PCR mutagenesis was used tomutate the glutamine (Q) at position 1 (according to Kabat numbering,see FIG. 1) of the variable heavy chain of humanized anti-Factor D Fabantibody variant 238 to a glutamate (E), resulting in humanizedanti-Factor D Fab antibody variant 238-1 (also herein referred to as“238-1”). The construction of the plasmid for humanized anti-Factor DFab antibody variant 238-1 (plasmid is herein referred to as “p238-1”)involved the ligation of two DNA fragments. The first fragment was thep238 vector in which the small BsiWI/PspOMI fragment had been removed.The second fragment was an approximately 364 base pair BsiWI-PspOMI PCRfragment generated from the p238 plasmid, using the following primers:

(SEQ ID NO: 71) 5′-TTTGGGTTTCGTACGCTGAAGTCCAGCTGGTGCAATCTGGG-3′ (SEQ IDNO: 72) 5′-TTTGGGTTTGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCAGGG T-3′.This ligation of the two DNA fragments resulted in the plasmid forhumanized anti-Factor D Fab antibody variant 238-1 (also herein referredto as “238-1”; plasmid is herein referred to as “p238-1”), whichincluded the site-directed mutation of the position 1 to a glutamate(E). This mutation was found to inhibit the partial conversion of theglutamine (Q) in humanized anti-Factor D Fab antibody variant 238-1 topyroglutamate (Amphlett, G. et al., Pharm. Biotechnol., 9:1-140 (1996)).

Further site-directed PCR mutagenesis may be used to mutate methionine(M or Met) or tryptophan (W or Trp) residues to prevent oxidation or tomutate asparagine (N or Asn) residues to prevent deamidation. To preventthe formation of oxidized variants of the humanized anti-Factor Dantibodies, methionines (M or Met), for example at position 33 of thelight chain may be mutated to leucine (L or Leu) which is most similarin size and hydrophobicity to methionine, but lacks a sulfur foroxidation, or alternatively mutated to isoleucine (I or Ile) (Amphlett,G. et al., Pharm. Biotechnol., 9:1-140 (1996)). To prevent the formationof deamidated variants of the humanized anti-Factor D antibodies,asparagines (N or Asn), for example at position 34 and 52 of the lightchain or position 99 or 100 of the heavy chain may be mutated toglutamine (Q or Gln) which is most similar chemically to asparagine (Nor Asn), or mutated to alanine (A or Ala) or serine (S or Ser) which arecommon substitutions at those positions in other antibodies (Amphlett,G. et al., Pharm. Biotechnol., 9:1-140 (1996)).

FIGS. 1-2 shows the variable light chain domain and variable heavy chaindomain sequences for humanized anti-Factor D Fab antibody variant 238(SEQ ID Nos: 6 and 18, respectively) and humanized anti-Factor D Fabantibody variant 238-1 (SEQ ID NOs: 7 and 19, respectively). FIG. 4 andFIG. 6 show the light chain and heavy chain sequences (SEQ ID NOs: 47and 54, respectively) for humanized anti-Factor D Fab antibody variant238. FIG. 8 and FIG. 10 show the light chain and heavy chain sequences(SEQ ID NOs: 61 and 63, respectively) for humanized anti-Factor D Fabantibody variant 238-1.

BiaCore data showed affinity of humanized anti-Factor D Fab antibodyvariant 238 to human Factor D as well as affinity of humanizedanti-Factor D full-length mAb version of clone #111 (humanizedanti-Factor D full-length mAb 234) (herein referred to as “234” or“anti-Factor D full-length mAb 234” or “humanized anti-Factor Dfull-length MAb 234”) (Table 2). Humanized anti-Factor D Fab antibodyvariants 238 and 238-1 were also tested in the hemolytic inhibitionassay (FIG. 11) to assess the inhibition of the alternative pathway (seeExample 3).

Example 2 AP Hemolysis Assay

Biological function of modified anti-Factor D Abs were determined usinghemolytic inhibition assay using C1q-depleted human serum and BiaCoreanalysis (See Example 3 below). Hemolytic assay was performed asfollows.

For determining alternative pathway activity, rabbit erythrocytes (Er,Colorado Serum) were washed 3× in GVB and resuspended to 2×10⁹/ml.Inhibitors (50 μl) and 20 μl of Er suspension were mixed 1:1 withGVB/0.1 M EGTA/0.1 M MgCl₂. Complement activation was initiated by theaddition of C1q-depleted human serum (to avoid any complement activationthrough the classical pathway) (CompTech; 30 μl diluted 1:3 in GVB).After a 30 minute incubation at room temperature, 200 μl GVB/10 mM EDTAwere added to stop the reaction and samples were centrifuged for 5 minat 500 g. Hemolysis was determined in 200 μl supernatant by measuringabsorbance at 412 nm. Data were expressed as % of hemolysis induced inthe absence of the inhibitor.

FIG. 11 shows inhibition of humanized anti-Factor D Fab clone #111(IC₅₀=4.7±0.6 nM), modified anti-Factor D Fab 238 (IC₅₀=6.4+0.6 nM) andmodified anti-Factor D Fab 238-1 (IC₅₀=3.5+0.5 nM) on alternativepathway hemolysis using rabbit red blood cell hemolysis assay usingC1q-depleted human serum as complement source. Controls were Factor H(“Human fH”) (from CompTech) and anti-glycoprotein 120 (“xgp120”)antibodies.

Example 3 Kinetic Analysis of anti-Human Factor D Fab by BiaCore

Kinetic and affinity constants for binding of human Factor D (AdvancedResearch, Inc.) to immobilized modified anti-factor D Fab 238 (hereinreferred to as “238”; see Example 1) were determined by surface plasmonresonance measurements on both BiaCore 3000 and BiaCore A100instruments. For the values in Table 2 that are listed as “BiaCore3000/BiaCore A100” results, separate experiments were done on eachinstrument, the data from the separate experiments were analyzed to getkinetic constants, and the kinetic constants were averaged to get thevalues shown in Table 2. Alternatively, kinetic and affinity constantsfor binding of human Factor D may be measured by immobilizing humanFactor D and measuring the binding of the mAb or Fab, may be measuredusing different regeneration conditions (e.g. comprising 4M MgCl₂)and/or may be measured using different binding buffers (e.g. comprisingPBS). Humanized anti-factor D full-length mAb version of clone #111(herein referred to as “234” or “anti-Factor D full-length mAb 234” or“humanized anti-Factor D full-length mAb 234”) was also analyzed.

1. Immobilization

mAb or Fab were immobilized via amine-coupling using a standard protocolsupplied by the manufacturer. The density of the coupling was regulatedby adjusting the concentration or pH of the injected mAb or Fabsolutions such that the total signal for saturating binding of humanfactor D was between 50 and 150 resonance units (RU). After coupling ofthe desired amount of mAb or Fab, unreacted functional groups on thesensor chip were blocked by injection of ethanolamine.

2. Kinetic Analysis

Binding experiments were conducting by injecting 60 μL aliquots of aseries of human factor D solutions varied in concentration from 500 nMto 0.98 nM in 2-fold increments. All samples were diluted in runningbuffer composed of 150 mM NaCl, 0.01% Tween-20 and one of the followingbuffer components: (a) pH 7.2 (10 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); (b) pH 6.0 (10 mMMES (2-[N-Morpholino]ethanesulfonic acid); or pH 5.0 (10 mM sodiumacetate). The flow rate was 30 μL/min and dissociation was monitored for10 minutes for each concentration of human factor D tested. The signal(sensorgram) observed for injection of the same solutions over areference cell (ethanolamine blocked) was subtracted from thesensorgram. Between sensorgrams the surface was regenerated by injectionof 30 μL of 4 M MgCl₂ to cause dissociation of any human factor Dremaining bound to the immobilized antibody. A control sensorgramrecorded for injection of buffer only over the sensor chip surface wassubtracted from the human factor D sensorgrams. These data were analyzedby non-linear regression according to a 1:1 Langmuir binding model usingBIAevaluation software v4.1. Kinetic and affinity constants are providedin the Table 2 below. BiaCore technology is limited and is not able toaccurately measure on-rates that are too fast (i.e. K_(D) values smallerthan about 10 pM) (Safsten et al., Anal. Biochem., 353: 181 (2006)).

TABLE 2 BiaCore Results Fab or Antibody ka (M−1s−1) kd (s−1) K_(D) (M)Modified Anti-factor D Fab 238 1.5 × 10⁸ 1.7 × 10⁻⁴  1.0 × 10⁻¹² (pH7.2; A100) (1.0 pM ± 0.05) Modified Anti-factor D Fab 238 8.4 ± 9 × 10⁸1.4 ± 1.7 × 10⁻³  1.4 × 10⁻¹² (pH 7.2; 3000/A100) (1.4 pM ± 0.5)Modified Anti-factor D Fab 238 1.9 × 10⁶ 3.6 × 10⁻⁴ 0.19 × 10⁻⁹ (pH 6;3000) (0.19 nM ± 0.01) Modified Anti-factor D Fab 238 1.2 × 10⁶ 0.0212.3 × 10⁻⁹ (pH 5; A100) (12.3 nM ± 2) Anti-factor D full-length mAb 2341.9 × 10⁸ 1.3 × 10⁻⁴  0.7 × 10⁻¹² (pH 7.2; A100) (0.7 pM ± 0.04)Anti-factor D full-length mAb 234 9.5 ± 10 × 10⁸ 1.3 ± 1.7 × 10⁻³  1.1 ×10⁻¹² (pH 7.2; 3000/A100) (1.1 pM ± 0.6) Anti-factor D full-length mAb234 2.8 × 10⁶ 2.2 × 10⁻⁴  .08 × 10⁻⁹ (pH 6; 3000) (0.08 nM ± 0.01)Anti-factor D full-length mAb 234 2.2 × 10⁶ 2.0 × 10⁻²   9 × 10⁻⁹ (pH 5;A100) (9.0 nM ± 1.0)

Example 4 AP Hemolysis Assay with Varying Factor D Concentrations

Biological function of modified anti-Factor D Abs, including anti-FactorD Fab 238, were determined using hemolytic inhibition assay usingC1q-depleted human serum and BiaCore analysis (See Example 2 above), inthe presence of three serum concentrations of Factor D.

C1q-depleted human serum (CompTech) as well as vitreous fluid andBruch's membrane tissue from eyes of AMD patients (obtained through acollaboration with the Cole Eye Institute, Cleveland, Ohio) wereanalyzed in a quantitative ELISA for Factor D (see below). Theconcentration of Factor D in the C1q-depleted serum was 97 nM, whereasthe level in vitreous fluid and Bruch's membrane tissue from AMDpatients was 16.2±10.3 nM (mean±SD, n=10).

The quantitative factor D ELISA was performed by diluting anti-humancomplement factor D goat polyclonal antibody (R&D Systems, Minneapolis,Minn.) to 1 μg/mL in phosphate buffered saline (PBS) and coating theanti-factor D polyclonal antibody (R&D Systems, Minneapolis, Minn.) onto384 well ELISA plates (high-bind plates; Greiner Bio One through VWRInternational, Bridgeport, N.J.) during an overnight incubation at 4° C.After washing 3 times with wash buffer (PBS/0.05% Tween-20), the plateswere blocked for 1-2 hr with PBS/0.5% bovine serum albumin (BSA). Thisand all other incubations were performed at room temperature on anorbital shaker. A standard curve of factor D (Complement Technology,Inc., Tyler, Tex.) was prepared in PBS/0.5% BSA/0.05% Tween-20 over arange of 15.6-1,000 μg/ml. Frozen control samples pre-diluted toquantitate at the high, mid, and low regions of the standard curve werethawed. C1q-depleted human serum and human vitreous fluid and Bruch'smembrane lysate samples were diluted using Assay Diluent (PBS/0.5%BSA/0.5% Tween-20). After the blocking step, the plates were washed andthe diluted samples (serum, vitreous fluid, and lysates of Bruch'smembrane), standards and controls were added and incubated for 2 hours.After the 2 hr incubation, the plates were washed, and bound factor Dwas detected during a 1 to 2 hr incubation with a biotinylatedanti-factor D monoclonal antibody (clone 9G7.1.16, produced atGenentech, diluted to 62.5 ng/ml) followed by a 30 min incubation withstreptavidin-horseradish peroxidase (SA-HRP)_(Amersham PharmaciaBiotech, Piscataway, N.J.), diluted 1/10,000 in Assay Diluent. Followinga final wash step, tetramethyl benzidine (Kirkegaard & PerryLaboratories, Inc., Gaithersburg, Md.) was added, and color was allowedto develop for 5 to 7 min. The reaction was stopped by the addition of 1M phosphoric acid. The optical density was read using a microplatereader (450 nm, 650 nm reference), and the sample concentrations werecalculated from four-parameter fits of the standard curves. The minimumquantifiable concentrations of factor D in human vitreous fluid andBruch's membrane lysate samples were 780 pg/mL (1/50 minimum dilution)and 156 μg/mL ( 1/10 minimum dilution), respectively.

In order to determine the IC₅₀ and IC₉₀ values for inhibition of thealternative complement pathway using modified anti-Factor D Fab 238 inthe presence of factor D concentrations similar to the concentrations ofFactor D observed in vitreous fluid and Bruch's membrane tissues fromeyes of AMD patients, the hemolytic assay was performed as described inExample 2, using 10% C1q-depleted serum (9.7 nM factor D) or using 10%C1q-depleted serum supplemented with additional factor D (CompTech) toachieve factor D concentrations representing the mean (16.2 nM) or themean±1 SD (26.5 nM) concentration observed in vitreous fluid and Bruch'smembrane tissues from eyes of AMD patients. Data were expressed as % ofhemolysis induced in the absence of the inhibitor (FIG. 12). Theconcentrations of anti-Factor D Fab 238 causing 50% and 90% inhibitionof the hemolytic reaction (IC₅₀ and IC₉₀ values, respectively) weredetermined for three repeat experiments by non-linear regression of theinhibition curves using a four-parameter fit model (KaleidaGraph,Synergy Software, Reading, Pa.). The molar ratios of the IC₅₀ and IC₉₀values versus the relative concentration of Factor D were alsocalculated. The average IC₅₀ and IC₉₀ values and molar ratios are shownin Table 3.

TABLE 3 IC50 and IC90 for Anti-Factor D Fab Anti-Factor D Fab (238-1)IC50 IC90 Factor D Molar Molar Concentration ratio Ratio (nM) nM(IC₅₀/fD) nM (IC₉₀/fD)  9.7 (nM)  4.4 ± 1.5 0.454 14.0 ± 1.0 1.443 16.2(nM) 10.2 ± 0.8 0.630  38.0 ± 11.0 2.346 26.5 (nM) 23.9 ± 5.0 0.902 72.6± 4.8 2.740

Example 5 Duration of Inhibition of Alternative Pathway ComplementActivation

The simulated duration of inhibition of the alternative pathway (AP)complement activation in a human eye using a single intravitreal (IVT)injection of anti-Factor D Fab 238 at a 2.5 mg dose (assuming ahalf-life (t_(1/2)) of anti-Factor D Fab 238 of 11.5 days, based oninterspecies scaling from the rabbit), was measured (Example 13). Thesimulated data are based on scaling from a PK study of singleintravitreal dose of Fab 238 in the New Zealand white rabbit.

To estimate the half-life of anti-Factor D Fab 238 in humans, thehalf-life of anti-Factor D 238 in rabbits was calculated. Twelve (12)New Zealand White rabbits were administered a single intravitreal doseof 1 mg Fab 238 in each eye. Vitreous humor and retinal tissue sampleswere collected from both eyes from the specified number of animals atthe following timepoints; 3 animals at 4, 24 and 96 hours (n=6 samplesat each of these timepoints) and one animal at 216 hours (n=2 samples atthis timepoint) and 2 animals at 240 hours (n=4 samples at thistimepoint). The concentrations of Fab 238 in the ocular matrices weremeasured in a factor D binding ELISA.

The vitreous humor concentration-time data from all animals wereanalyzed to estimate pharmacokinetic parameter estimates using a naïvepooled approach with the IV bolus input model (Models 201, WinNonlin Proversion 5.2.1; Pharsight Corporation, Mountain View, Calif.) to provideone estimate of terminal half-life (T_(1/2)) of 3.83 days. The retinalpartition coefficient was calculated as the ratio of the concentrationin the retinal tissue to vitreous humor averaged for all eyes at alltimepoints, and was equal to 0.24. The PK parameters for vitreous humorwere scaled to human using the same scaling factors observed forranibizumab. The human eye is assumed to have a V₁ of 4 mL, the ratio ofhalf-life in the human to the rabbit is assumed to be 3, producing anestimate of t_(1/2) in human of 11.5 days. This produced the estimatefor vitreous concentration and retinal tissue concentrations as afunction of time as:Vitreous Concentration=(Dose/V ₁)*exp([−In(2)/t _(1/2)]*time)Retinal tissue Concentration=(Dose/V ₁)*exp([−In(2)/t_(1/2)]*time)*(retinal partition coefficient)

In FIG. 13, the graph was produced for a single ITV dose of 2.5 mg/eye,and represents time from t=0 to t=112 days. IC₉₀ represents theconcentration of Fab 238 that produces a 90% inhibitory effect in thehemolysis assay performed as shown in Example 2 and 4 in which 10%pooled human serum was supplemented to a Factor D concentration of 16.2nM. The assay result was IC₉₀=38 nM Fab 238. To compare to the retinal &vitreous concentrations, a molar to mass conversion was done using thefollowing equation:IC ₉₀=38×10⁻⁹ moles/LMW of Fab 238=50,000 grams/moleIC ₉₀ (ug/mL)=(38×10⁻⁹ moles/L)×(50×10³ grams/mole)=1.9×10⁻⁹ grams/L, or1.9 ug/mL

As shown in FIG. 13, the “days above IC₉₀” was estimated as the amountof time the vitreous or retinal concentration would be predicted to beabove 1.9 ug/mL after a single ITV dose of 2.5 mg/eye, and was observedas the point where the graph of the vitreous or retinal concentrationscross the line at 1.9 ug/mL. A single IVT injection of anti-Factor D Fab238 was estimated to inhibit AP complement activation in the retinaltissue for at least about 74 days and in the vitreous humor for at leastabout 97 days. The dashed line in FIG. 13 shows the simulatedanti-Factor D Fab 238 concentration in the vitreous humor followingintravitreal administration. The solid line in FIG. 13 shows thesimulated anti-Factor D Fab 238 concentration in the retinal tissuefollowing intravitreal administration. The difference in theconcentration in the vitreous humor and retinal tissue is based upon anestimate of the retinal tissue partition coefficient of 20%; in otherwords, 20% of the total drug administered to the vitreous humor willhave access to the retinal tissue

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literatures cited herein are expressly incorporated in theirentirety by reference.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

1. An anti-Factor D antibody or an antigen-binding fragment thereofcomprising: (a) a light chain variable domain comprising: (1) lightchain HVR-1 (HVR-L1) comprising the sequence ITSTDIDDDMN (SEQ ID NO: 30)or a sequence that differs from SEQ ID NO: 30 by one or more amino acidsubstitutions, wherein said one or more substitutions are substitutionof the amino acid residue at position 10 of SEQ ID NO: 30 to L or Iand/or substitution of the amino acid at position 11 of SEQ ID NO: 30 toA or Q, (2) light chain HVR-2 (HVR-L2) comprising the sequence GGNTLRP(SEQ ID NO: 35) or a sequence that differs from SEQ ID NO: 35 by oneamino acid substitution, wherein said one substitution is substitutionof the amino acid residue at position 3 of SEQ ID NO: 35 to S or A, and,(3) light chain HVR-3 (HVR-L3) comprising the sequence LQSDSLPYT (SEQ IDNO: 38); and (b) a heavy chain variable domain comprising: (1) heavychain HVR-1 (HVR-H1) comprising the sequence GYTFTNYGMN (SEQ ID NO: 39),(2) heavy chain HVR-2 (HVR-H2) comprising the sequence WINTYTGETTYADDFKG(SEQ ID NO: 40), and (3) heavy chain HVR-3 (HVR-H3) comprising thesequence EGGVNN (SEQ ID NO: 41) or a sequence that differs from SEQ IDNO: 41 by one or more amino acid substitutions, wherein said one or moresubstitutions are substitution of the amino acid residue at position 5of SEQ ID NO: 41 with A or Q and/or substitution of the amino acidresidue at position 6 of SEQ ID NO: 41 with A or Q, wherein saidantibody or antigen-binding fragment thereof comprises at least one ofthe substitutions set forth in (a)(1), (a)(2), or (b)(3) above and/orthe heavy chain variable domain comprises an E at position
 1. 2. Theanti-Factor D antibody or antigen-binding fragment of claim 1, whereinthe anti-Factor D antibody or antigen-binding fragment thereof comprisesan E at position 1 of the heavy chain variable domain.
 3. Theanti-Factor D antibody or antigen-binding fragment of claim 2, whereinthe light chain variable domain comprises the amino acid sequence ofDIQVTQSPSSLSASVGDRVTITCITSTDIDDDX₄X₅WYQQKPGKVPKLLISGGX₆TLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDSLPYTFGQGTKX₇EIK (SEQ ID NO: 73),wherein X₄ is M, L or I; X₅ is N, A or Q; X₆ is N, S or A; and X₇ is Lor V.
 4. The anti-Factor D antibody or antigen-binding fragment of claim3, wherein X₇ is V.
 5. The anti-Factor D antibody or antigen-bindingfragment of claim 1, wherein (a) the heavy chain variable domaincomprises: i. an HVR-H1 comprising the amino acid sequence of SEQ ID NO:39; ii. an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 40;iii. an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41, SEQID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45; and (b) thelight chain variable domain comprises: iv. an HVR-L1 comprising theamino acid sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; v. an HVR-L2 comprising theamino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37;and vi. an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 38.6. The anti-Factor D antibody or antigen-binding fragment of claim 5,wherein the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 41.7. The anti-Factor D antibody or antigen-binding fragment of claim 5,wherein the HVR-L1 comprises the amino acid sequence of SEQ ID NO: 30.8. The anti-Factor D antibody or antigen-binding fragment of claim 5,wherein the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 35.9. The anti-Factor D antibody or antigen-binding fragment of claim 5,wherein the HVR-L1 comprises the amino acid sequence of SEQ ID NO: 30,the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 35, and theHVR-L3 comprises the amino acid sequence of SEQ ID NO:
 38. 10. Theanti-Factor D antibody or antigen-binding fragment of claim 5, whereinthe HVR-H1 comprises the amino acid sequence of SEQ ID NO: 39, theHVR-H2 comprises the amino acid sequence of SEQ ID NO: 40, the HVR-H3comprises the amino acid sequence of SEQ ID NO: 41, the HVR-L1 comprisesthe amino acid sequence of SEQ ID NO: 30, the HVR-L2 comprises the aminoacid sequence of SEQ ID NO: 35, and the HVR-L3 comprises the amino acidsequence of SEQ ID NO:
 38. 11. The anti-Factor D antibody orantigen-binding fragment of claim 10, wherein the light chain variabledomain comprises the amino acid sequence of SEQ ID NO: 1, and whereinthe amino acid residue at position 104 of SEQ ID NO: 1 is substitutedwith V.
 12. The anti-Factor D antibody or antigen-binding fragment ofclaim 11, wherein the amino acid residue at position 1 of the heavychain variable domain is an E.
 13. The anti-Factor D antibody orantigen-binding fragment of claim 1, wherein the heavy chain variabledomain comprises the sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO:
 29. 14. Theanti-Factor D antibody or antigen-binding fragment of claim 1, whereinthe light chain variable domain comprises the sequence of SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, or SEQ ID NO:
 14. 15. An anti-Factor D antibody orantigen-binding fragment thereof, comprising a polypeptide comprisingthe following amino acid sequence:X₁VQLVQSGPELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGETTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCEREGGVX₂X₃WGQGTLVTVS S (SEQ IDNO: 74), wherein X₁ is E; X₂ is N, A or Q; and X₃ is N, A or Q.
 16. Theanti-Factor D antibody or antigen-binding fragment of claim 15, whereinthe anti-Factor D antibody or antigen-binding fragment thereof furthercomprises a polypeptide comprising the following amino acid sequence:DIQVTQSPSSLSASVGDRVTITCITSTDIDDDX₄X₅WYQQKPGKVPKLLISGGX₆TLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDSLPYTFGQGTKX₇EIK (SEQ ID NO:73),wherein X₄ is M, L or I; X₅ is N, A or Q; X₆ is N, S or A; and X₇ is Lor V.
 17. An anti-Factor D antibody or an antigen-binding fragmentthereof, comprising the heavy chain variable domain sequence of SEQ IDNO:
 19. 18. The anti-Factor D antibody or antigen-binding fragment ofclaim 17, wherein the anti-Factor D antibody or antibody fragmentthereof further comprises the light chain variable domain sequence ofSEQ ID NO:
 7. 19. An anti-Factor D antibody or an antigen-bindingfragment thereof, comprising the heavy chain sequence of SEQ ID NO: 63.20. The anti-Factor D antibody or antigen-binding fragment of claim 19,wherein the anti-Factor D antibody further comprises the light chainsequence of SEQ ID NO:
 61. 21. An anti-Factor D antibody or anantigen-binding fragment thereof, comprising: (a) a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 1, wherein theamino acid residue at position 104 of SEQ ID NO: 1 is substituted withV; and (b) a heavy chain variable domain comprising an HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 39, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 40, and an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 41, wherein the amino acid residue at position 1of the heavy chain variable domain is an E.
 22. An anti-Factor Dantibody or an antigen-binding fragment thereof made by the process of:(a) culturing a cell expressing an anti-Factor D antibody or anantigen-binding fragment thereof comprising the heavy chain variabledomain sequence of SEQ ID NO: 19 and the light chain variable domainsequence of SEQ ID NO: 7; and (b) isolating the antibody orantigen-binding fragment from said cultured cell.
 23. The anti-Factor Dantibody or antigen-binding fragment of any one of claims 1, 15, 16, 17,18 and 21, wherein the anti-Factor D antibody is a monoclonal antibody.24. The anti-Factor D antibody or antigen-binding fragment of any one ofclaims 1, 15, 16, 17, 18 and 21, wherein the antigen-binding fragment isa Fab, Fab′-SH, Fv, scFv, or (Fab′)₂ fragment.
 25. The anti-Factor Dantibody or antigen-binding fragment of any one of claims 1, 15, 16, 17,18 and 21, wherein the anti-Factor D antibody is a humanized antibody.26. The anti-Factor D antibody or antigen-binding fragment of any one ofclaims 1, 15, 16, 17, 18, 19, 20 and 21, wherein the Factor D ismammalian Factor D.
 27. The anti-Factor D antibody or antigen-bindingfragment of claim 26, wherein the Factor D is human Factor D.
 28. Theanti-Factor D antibody or antigen-binding fragment of any one of claims1, 15, 16, 17, 18, 19, 20 and 21, which is produced in bacteria.
 29. Theanti-Factor D antibody or antigen-binding fragment of any one of claims1, 15, 16, 17, 18, 19, 20 and 21, which is produced in CHO cells.
 30. Apharmaceutical formulation comprising the anti-Factor D antibody orantigen-binding fragment of any one of claims 1, 15, 16, 17, 18, 19, 20and 21, and a pharmaceutically acceptable diluent, carrier or excipient.31. An article of manufacture comprising: (a) the pharmaceuticalformulation of claim 30; (b) a container; and (c) a package insert orlabel indicating that the pharmaceutical formulation-can be used totreat a complement-associated disorder.
 32. A kit comprising theanti-Factor D antibody or antigen-binding fragment of any one of claims1, 15, 16, 17, 18, 19, 20 and 21, and instructions for administeringsaid antibody to treat a complement-associated disorder.
 33. The kit ofclaim 32, wherein the complement-associated disorder is an oculardisease.
 34. The kit of claim 33, wherein the ocular disease is selectedfrom the group consisting of age-related macular degeneration (AMD),diabetic retinopathy, choroidal neovascularization (CNV), uveitis,diabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO),corneal neovascularization, and retinal neovascularization.
 35. The kitof claim 34, wherein the age-related macular degeneration isintermediate dry AMD or geographic atrophy.